Neuroprotective agents derived from spider venom peptides

ABSTRACT

The invention relates to disulfide-rich peptides derived from spider venom and their use, particularly as neuroprotective agents. The invention also relates to nucleic acid molecules encoding the peptides as well as constructs and host cells comprising those nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/745,432, 371(c) date Jan. 16, 2018, which is the U.S. national phaseof International Application No. PCT/AU2016/050633, filed Jul. 18, 2016,which designated the U.S. and claims priority to Australian PatentApplication No. 2015902845, filed Jul. 17, 2015; the entire contents ofeach of which are hereby incorporated by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:0181_0444_amended_Sequence_Listing.TXT, Size: 34,547 bytes; and Date ofCreation: Oct. 31, 2019) is herein incorporated by reference in itsentirety.

TECHNICAL FIELD

The present invention relates to disulfide-rich peptides derived fromspider venom and their use, particularly as neuroprotective agents. Moreparticularly, in one aspect, the invention relates to disulfide-richpeptides that have neuroprotective activity following stroke as well asto compositions containing such peptides and their use in the treatmentof stroke. The invention also relates to nucleic acid molecules encodingthe peptides as well as constructs and host cells comprising thosenucleic acid molecules.

BACKGROUND ART

Stroke is the second leading cause of death worldwide (Woodruff et al.,2011; Moskowitz et al., 2010), and the third leading cause of mortalityin Australia (Senes, 2006). In addition to the high rate of mortality,there is an extremely high incidence of morbidity in stroke survivors,making it the leading cause of disability in industrialised countries(Liu et al., 2012).

Within a few minutes of the onset of cerebral ischemia, the infarct coreis mortally injured and undergoes necrotic cell death. The infarct core(or striatal region) of the stroke is commonly considered unsalvageable.The necrotic core is surrounded by a zone of less severely affectedtissue known as the ischemic penumbra or peri-infarct zone, which ispotentially salvageable via post-stroke therapy (Woodruff et al., 2011).The use of recombinant tissue plasminogen activator (rtPA) to helprestore blood flow to the ischemic region is, to date, the only approvedagent for treatment of acute ischemic stroke. It is used in only 3-4% ofall stroke patients (Besancon et al., 2008) due to its narrowtherapeutic window and the risk of inducing intracranial haemorrhage(Moskowitz et al., 2010). There is clearly a need for more effectiveneuroprotective agents. A longer time window for therapeuticintervention would also be advantageous.

During cerebral ischemia, severe oxygen depletion occurs, compelling thebrain to switch from oxidative phosphorylation to anaerobic glycolysis,leading to acidosis as a result of increased lactate levels. Theextracellular pH can fall from ˜7.3 to 6.0-6.5 in the ischemic coreunder normoglycemic conditions, and can drop to below 6.0 during severeischemia (Xiong et al., 2004; Isaev et al., 2008). The drop inextracellular pH activates acid sensing ion channels (ASICs), andactivation of these channels is thought to play a critical role instroke-induced neuronal injury. Numerous studies have demonstrated adirect correlation between brain acidosis and infarct size (Xiong etal., 2007).

ASICs were discovered in the late 1990s, almost 20 years after theobservation that sensory neurons depolarise in response to a sudden dropin pH (Krishtal, 2003). Although they belong to the epithelial sodiumchannel/degenerin family of receptors, they are distinguished by theirrestriction to chordates, predominantly neuronal distribution, andactivation by decreases in extracellular pH (Gründer and Chen, 2010).Alternative splicing of four ASIC-encoding genes leads to the expressionof six subunits (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4) thatcombine to form hetero- or homo-trimeric channels that differ in theirpH sensitivity, kinetics, and susceptibility to desensitisation (Wemmieet al., 2006).

Postsynaptic ASIC1a channels are the dominant ASIC subtype in mammalianbrain (Xiong et al., 2004; Li et al., 2010). The pH for half-maximalactivation (pH_(0.5)) of ASIC1a is 6.6 in human cortical neurons (Li etal., 2010) and 6.4 in rat Purkinje neurons, and consequently they arerobustly activated by the decrease in extracellular pH that occursduring cerebral ischemia. Importantly, homomeric ASIC1a channels canmediate the uptake of Ca²⁺ in addition to Na⁺ and protons (Gründer andChen, 2010). Thus, brain ASIC1a can contribute to the intracellular Ca²⁺overload that occurs during stroke, and the proton permeability ofASIC1a may be at least partly responsible for the precipitous drop inintracellular pH from ˜7 to as low as 6.15 during cerebral ischemia(Isaev et al., 2008).

It is now known that cerebral acidosis activates ASIC1a and that thisactivation is a major contributor to the neuronal damage resulting fromstroke (Xiong et al., 2004; Xiong et al., 2007; Wang et al., 2011; Lenget al., 2013). For example, in rodent models of cerebral ischemia,infarct size and neurological deficits are greatly reduced by knockoutor pharmacological blockade of ASIC1a (Xiong et al., 2004).

The most potent and selective blocker of ASIC1a described to date isPcTx1 (also known as π-theraphotoxin-Pc1a), a 40-residue peptideisolated from the venom of the Trinidad Chevron tarantula, Psalmopoeuscambridgei. PcTx1 inhibits rat ASIC1a (rASIC1a) with an IC₅₀ of ˜1 nMbut it does not inhibit other ASIC homomers or heteromers. In a ratmodel of transient focal ischemia (middle cerebral artery occlusion;MCAO), intracerebroventricular (i.c.v.) injection of P. cambridgei crudevenom (that is, without isolating the pure ASIC1a inhibitory peptide)reduced infarct size by 60%. Consistent with this being an effectmediated by ASIC1a, infarct size was similarly reduced by 61% inASIC1^(−/−) mice (Xiong et al., 2004). Also, infarct size was reduced by˜30% when P. cambridgei crude venom was delivered i.c.v. as late as fivehours after MCAO (Pignataro et al., 2007).

Whilst PcTx1 appears to be a promising lead molecule for the developmentof neuroprotective agents for the treatment of stroke, there remains asignificant need for compounds that can form the basis of an effectiveneuroprotective therapy. Given the delay in clinical presentationfollowing stroke, a longer time window for therapeutic interventionwould also be advantageous.

It will be clearly understood that, if a prior art publication isreferred to herein, this reference does not constitute an admission thatthe publication forms part of the common general knowledge in the art inAustralia or in any other country.

SUMMARY OF INVENTION

The invention is broadly directed to disulfide-rich peptides derivedfrom spider venom and their use as neuroprotective agents, particularlyin the treatment of stroke.

In a first aspect, the present invention provides an isolated, syntheticor recombinant disulfide-rich peptide with neuroprotective activity. Thepeptide comprises, consists of, or consists essentially of, a sequenceof Formula (I):

X-L-Y   (I)

wherein X and Y each represent a peptide sequence having an inhibitorcystine knot (ICK) fold and L is a linker, and wherein said peptide ispreferably capable of specifically binding to acid sensing ion channelsubtype 1a (ASIC1a).

In a second aspect, the present invention provides a functionally activeneuroprotective fragment, derivative or analogue of the disulfide-richpeptide provided by the first aspect that is preferably capable ofspecifically binding to ASIC1a.

In a third aspect, the present invention provides a neuroprotectivepeptide in which two ICK motifs are joined head-to-tail by a six-residuelinker, and wherein said peptide is preferably capable of specificallybinding to ASIC1a.

In a fourth aspect, the present invention provides a neuroprotectivepeptide comprising twelve cysteine residues covalently joined in pairsto form six disulfide bonds, such that the peptide comprises two ICKmotifs, and wherein said peptide is preferably capable of specificallybinding to ASIC1a.

In a fifth aspect, the present invention provides an isolated, syntheticor recombinant disulfide-rich peptide with neuroprotective activity,wherein the peptide comprises, consists of, or consists essentially of,an amino acid sequence selected from the group consisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8,        10, 12, 14 or 16;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 or 16;    -   (c) a sequence that is encoded by the nucleotide sequence set        forth in any one of SEQ ID NOs:1, 3, 5, 7, 9, 11 or 13;    -   (d) a sequence that is encoded by a nucleotide sequence that        shares at least 65% (and at least 66% to at least 99% and all        integer percentages in between) sequence similarity or sequence        identity with the sequence set forth in any one of SEQ ID NOs:        1, 3, 5, 7, 9, 11 or 13; or    -   (e) a sequence that is encoded by a nucleotide sequence that        hybridizes under at least medium or high stringency conditions        to the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13.

In a sixth aspect, the present invention provides an isolated, syntheticor recombinant disulfide-rich peptide derived from spider venom that iscapable of specifically binding to ASIC1 a.

In a seventh aspect, the present invention provides an isolated,synthetic or recombinant nucleic acid molecule that comprises, consistsof, or consists essentially of, a nucleotide sequence encoding the aminoacid sequence of the disulfide-rich peptide provided by the first orsixth aspects, the fragment, derivative or analogue provided by thesecond aspect, or the neuroprotective peptide provided by the third orfourth aspects. In some embodiments, the nucleic acid moleculecomprises, consists of, or consists essentially of, a nucleotidesequence selected from the group consisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 or 13, or a complement        thereof; or    -   (c) a sequence that hybridizes under at least medium or high        stringency conditions to the sequence set forth in any one of        SEQ ID NOs:1, 3, 5, 7, 9, 11 or 11, or a complement thereof.

In an eighth aspect, the present invention provides a genetic constructfor expressing the nucleic acid molecule provided by the seventh aspect(for example, for making recombinant peptides in commercial quantities).The genetic construct generally comprises the isolated nucleic acidmolecule provided by the seventh aspect operably linked to one or moreregulatory sequences in an expression vector.

In a ninth aspect, the present invention provides a host celltransformed with the nucleic acid molecule provided by the seventhaspect or the genetic construct provided by the eighth aspect.

In a tenth aspect, the present invention provides a method of producingthe isolated, synthetic or recombinant disulfide-rich peptide providedby the first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects, comprising culturing the transformedhost cell provided by the ninth aspect and isolating the resultantpeptide, fragment, derivative or analogue from said cultured host cell.

In an eleventh aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone isolated, synthetic or recombinant disulfide-rich peptide providedby the first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects.

In a twelfth aspect, the invention provides a method for the treatmentof stroke in a subject comprising the step of administering atherapeutically effective amount of at least one isolated, synthetic orrecombinant disulfide-rich peptide provided by the first or sixthaspects, the fragment, derivative or analogue provided by the secondaspect, the neuroprotective peptide provided by the third or fourthaspects or the pharmaceutical composition provided by the eleventhaspect.

In a thirteenth aspect, the invention provides a method for theprevention or treatment of neuronal damage following stroke in a subjectcomprising the step of administering a therapeutically effective amountof at least one isolated, synthetic or recombinant disulfide-richpeptide provided by the first or sixth aspects, the fragment, derivativeor analogue provided by the second aspect, the neuroprotective peptideprovided by the third or fourth aspects or the pharmaceuticalcomposition provided by the eleventh aspect.

In a fourteenth aspect, the invention provides a method of reducinginfarct size following stroke in a subject comprising the step ofadministering a therapeutically effective amount of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, the neuroprotective peptide provided bythe third or fourth aspects or the pharmaceutical composition providedby the eleventh aspect.

In a fifteenth aspect, the invention provides a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the first or sixth aspects, thefragment, derivative or analogue provided by the second aspect, theneuroprotective peptide provided by the third or fourth aspects or thepharmaceutical composition provided by the eleventh aspect for thetreatment of stroke.

In a sixteenth aspect, the invention provides a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the first or sixth aspects, thefragment, derivative or analogue provided by the second aspect, theneuroprotective peptide provided by the third or fourth aspects or thepharmaceutical composition provided by the eleventh aspect for theprevention or treatment of neuronal damage following stroke in asubject.

In a seventeenth aspect, the invention provides a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the first or sixth aspects, thefragment, derivative or analogue provided by the second aspect, theneuroprotective peptide provided by the third or fourth aspects or thepharmaceutical composition provided by the eleventh aspect for thereduction of infarct size following stroke in a subject.

In an eighteenth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects in the manufacture of a medicament forthe treatment of stroke.

In a nineteenth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects in the manufacture of a medicament forthe prevention or treatment of neuronal damage following stroke in asubject.

In a twentieth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects in the manufacture of a medicament forthe reduction of infarct size following stroke in a subject.

In a twenty-first aspect, the invention provides use of an isolated,synthetic or recombinant disulfide-rich neuroprotective peptide derivedfrom spider venom for treating stroke.

In a twenty-second aspect, there is provided a method for identifying ordesigning a peptide, peptidomimetic, or small molecule capable ofinhibiting activation of ASIC1a, said method comprising the steps of:

-   -   (i) computer modelling the interaction between ASIC1a and at        least one disulfide-rich peptide, wherein said disulfide-rich        peptide is as defined in any one of the first to sixth aspects;    -   (ii) using data generated by the computer modelling to identify        or design a peptide, peptidomimetic, or small molecule capable        of binding to ASIC1a and inhibiting the activation of ASIC1a;        and optionally,    -   (iii) producing the peptide, peptidomimetic, or small molecule        of step (ii), and optionally,    -   (iv) testing the peptide, peptidomimetic, or small molecule of        step (iii) for binding to ASIC1a and inhibiting the activation        of ASIC1a.

The invention is also more broadly directed to disulfide-rich peptidesderived from spider venom and their use as therapeutic or prophylacticagents. Such peptides can be used as therapeutic or prophylactic agentsfor conditions caused by ASIC1a activity or contributed to by ASIC1aactivity in biological pathways.

In a twenty-third aspect, the present invention provides an isolated,synthetic or recombinant disulfide-rich peptide. The peptide comprises,consists of, or consists essentially of, a sequence of Formula (I):

X-L-Y   (I)

wherein X and Y each represent a peptide sequence having an inhibitorcystine knot (ICK) fold and L is a linker, and wherein said peptide ispreferably capable of specifically binding to acid sensing ion channelsubtype 1a (ASIC1a).

In a twenty-fourth aspect, the present invention provides a functionallyactive fragment, derivative or analogue of the disulfide-rich peptideprovided by the twenty-third aspect that is preferably capable ofspecifically binding to ASIC1a.

In a twenty-fifth aspect, the present invention provides a peptide inwhich two ICK motifs are joined head-to-tail by a six-residue linker,and wherein said peptide is preferably capable of specifically bindingto ASIC1a.

In a twenty-sixth aspect, the present invention provides a peptidecomprising twelve cysteine residues covalently joined in pairs to formsix disulfide bonds, such that the peptide comprises two ICK motifs, andwherein said peptide is preferably capable of specifically binding toASIC1a.

In a twenty-seventh aspect, the present invention provides an isolated,synthetic or recombinant disulfide-rich peptide with therapeutic orprophylactic activity, wherein the peptide comprises, consists of, orconsists essentially of, an amino acid sequence selected from the groupconsisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8,        10, 12, 14 or 16;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 or 16;    -   (c) a sequence that is encoded by the nucleotide sequence set        forth in any one of SEQ ID NOs:1, 3, 5, 7, 9, 11 or 13;    -   (d) a sequence that is encoded by a nucleotide sequence that        shares at least 65% (and at least 66% to at least 99% and all        integer percentages in between) sequence similarity or sequence        identity with the sequence set forth in any one of SEQ ID NOs:        1, 3, 5, 7, 9, 11 or 13; or    -   (e) a sequence that is encoded by a nucleotide sequence that        hybridizes under at least medium or high stringency conditions        to the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13.

In a twenty-eighth aspect, the present invention provides an isolated,synthetic or recombinant disulfide-rich peptide derived from spidervenom that is capable of specifically binding to ASIC1a and inhibitingan ASIC1a biological pathway.

In a twenty-ninth aspect, the present invention provides an isolated,synthetic or recombinant nucleic acid molecule that comprises, consistsof, or consists essentially of, a nucleotide sequence encoding the aminoacid sequence of the disulfide-rich peptide provided by the twenty-thirdor twenty-eighth aspects, the fragment, derivative or analogue providedby the twenty-fourth aspect, or the peptide provided by the twenty-fifthor twenty-sixth aspects. In some embodiments, the nucleic acid moleculecomprises, consists of, or consists essentially of, a nucleotidesequence selected from the group consisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 or 13, or a complement        thereof; or    -   (c) a sequence that hybridizes under at least medium or high        stringency conditions to the sequence set forth in any one of        SEQ ID NOs:1, 3, 5, 7, 9, 11 or 11, or a complement thereof.

In a thirtieth aspect, the present invention provides a geneticconstruct for expressing the nucleic acid molecule provided by thetwenty-ninth aspect (for example, for making recombinant peptides incommercial quantities). The genetic construct generally comprises theisolated nucleic acid molecule provided by the twenty-ninth aspectoperably linked to one or more regulatory sequences in an expressionvector.

In a thirty-first aspect, the present invention provides a host celltransformed with the nucleic acid molecule provided by the twenty-ninthaspect or the genetic construct provided by the thirtieth aspect.

In a thirty-second aspect, the present invention provides a method ofproducing the isolated, synthetic or recombinant disulfide-rich peptideprovided by the twenty-third or twenty-eighth aspects, the fragment,derivative or analogue provided by the twenty-fourth aspect, or thepeptide provided by the twenty-fifth or twenty-sixth aspects, comprisingculturing the transformed host cell provided by the thirty-first aspectand isolating the resultant peptide, fragment, derivative or analoguefrom said cultured host cell.

In a thirty-third aspect, the present invention provides apharmaceutical composition comprising a therapeutically effective amountof at least one isolated, synthetic or recombinant disulfide-richpeptide provided by the twenty-third or twenty-eighth aspects, thefragment, derivative or analogue provided by the twenty-fourth aspect,or the peptide provided by the twenty-fifth or twenty-sixth aspects.

In a thirty-fourth aspect, the invention provides a method for theprevention or treatment of a condition in a subject caused by ASIC1aactivity or contributed to by ASIC1a activity comprising the step ofadministering a therapeutically effective amount of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe twenty-third or twenty-eighth aspects, the fragment, derivative oranalogue provided by the twenty-fourth aspect, the peptide provided bythe twenty-fifth or twenty-sixth aspects or the pharmaceuticalcomposition provided by the thirty-third aspect.

In a thirty-fifth aspect, the invention provides a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the twenty-third or twenty-eighthaspects, the fragment, derivative or analogue provided by thetwenty-fourth aspect, the peptide provided by the twenty-fifth ortwenty-sixth aspects or the pharmaceutical composition provided by thethirty-third aspect for the prevention or treatment of a condition in asubject caused by ASIC1a activity or contributed to by ASIC1a activity.

In a thirty-sixth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe twenty-first or twenty-eighth aspects, the fragment, derivative oranalogue provided by the twenty-fourth aspect, or the peptide providedby the twenty-fifth or twenty-sixth aspects in the manufacture of amedicament for the prevention or treatment of a condition in a subjectcaused by ASIC1a activity or contributed to by ASIC1a activity.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

Any of the features described herein can be combined in any combinationwith any one or more of the other features described herein within thescope of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Alignment of the amino acid sequences of members of theπ-hexatoxin-Hi1a (Hi1a) superfamily (Hi1a—SEQ ID NO:16; Hi1b—SEQ IDNO:29; Hi1c—SEQ ID NO:30; Hi1d—SEQ ID NO: 31; Hi1e—SEQ ID NO: 32).Cysteine residues are highlighted in white text on a black background.Residues that differ from those found at the corresponding position inHi1a are highlighted in black text on a grey background. The residuenumbers above the alignment refer to Hi1a.

FIG. 2. Characterisation of pure, recombinant Hi1a. (A) AnalyticalRP-HPLC chromatogram of pure recombinant Hi1a, which elutes as threepeaks with the same mass (re-injection of a single peak results again inthe appearance of three peaks). This is indicative of conformationalisomerism. The top and bottom traces correspond to absorbance at 214 nmand 280 nm, respectively. (B) Electrospray ionisation mass spectrometry(ESI-MS) spectrum of pure recombinant Hi1a. The average mass of 8723 Daindicates that the peptide contains the expected six disulfide bonds.

FIG. 3. Functional characterisation of recombinant Hi1a. (A)Concentration response curves for inhibition of rASIC1a and hASIC1a byHi1a (Note: data are mean±SEM; number of experiments=8). I/I_(max): testcurrent/control current. (B) Effect of 30 nM Hi1a at pH 7.45 on variousASIC subtypes and point mutants (Note: data are mean±SEM; number ofexperiments=5). (C) Effect of 1 nM and 1 μM Hi1a at pH 7.45 on variousASIC subtypes expressed in Xenopus oocytes. Individual data points areshown as well as mean±SEM; number of experiments=5. (D) Concentrationdependent effects of Hi1a on wild-type rASIC1a and an F350A mutantrASIC1a channel expressed in Xenopus oocytes.

FIG. 4. Representative recordings of experiments performed on Xenopusoocytes expressing rASIC1a channels. Whole-cell currents were elicitedby a drop in extracellular pH from 7.45 to 6.0, every 60 seconds (andevery 5 minutes during peptide washout). Oocytes expressing channelswere exposed to (A) PcTx1 and (B) Hi1a (at a constant concentration of10 nM) at pH 7.45 for 120 seconds twice. (C) Representative currenttraces in the absence (control) and presence of Hi1a (0.1 nM and 1 μM)showing incomplete current inhibition with 1 μM Hi1a (saturatingconcentration). (D) Graph of ASIC1a current versus time after peptidewashout showing the rapid, fully reversible inhibition of ASIC1a byPcTx1 compared to the very slow reversibility of the inhibitory actionof Hi1a (˜40% recovery of current amplitude after 30 minutes of washout)(mean±SEM; number of experiments=5).

FIG. 5. Modulatory effect of Hi1a on ASIC1a steady-statedesensitisation. Effect of increasing concentrations of Hi1a on thesteady-state desensitisation (SSD) of rASIC1a. Control currents weretaken using a pH drop from 7.45 to pH 5, then the SSD curve was obtainedby conditioning the channel for 120 seconds at pH 7.9, 7.75, 7.6, 7.45,7.3, 7.2, 7.0 in the absence and presence of peptide (mean±SEM; numberof experiments=5).

FIG. 6. Modulatory effect of Hi1a on ASIC1a activation. Effect ofincreasing concentrations of Hi1a on the acid-evoked activation of (A)rASIC1a and (B) hASIC1a. The activation curves were obtained byconditioning the channel at pH 7.45 for 60 seconds in between increasingacidic stimuli from pH 7 to 5, in the absence and presence ofrecombinant peptide (mean±SEM; number of experiments=5).

FIG. 7. Effect of Hi1a on the pH-dependence of activation and SSD ofrASIC1a (left panel) and hASIC1a (right panel). The activation curveswere obtained by applying increasing concentrations of protons every 50seconds. In the continued presence of protons (pH values below ˜7.2 forrASIC1a), ASICs rapidly desensitise and cannot re-open untilsufficiently de-protonated (pH values >˜7.3 for rASIC1a). SSD profileswere obtained by conditioning the channels for 120 seconds at decreasingpH (mean±SEM; number of experiments=6). The symbols have the samemeaning in both panels: ●, no peptide; Δ, 0.5 nM Hi1a; □, 5 nM Hi1a.

FIG. 8. Structure of Hi1a. Schematic of the three-dimensional structureof Hi1a determined using nuclear magnetic resonance (NMR) spectroscopy(first structure in the ensemble of 20 structures in Protein Data Bankaccession 2N8F). The N- and C-terminal inhibitor cystine knot (ICK)domains are labelled as well as the interdomain linker. The labels “N”and “C” indicate the N- and C-terminus of the peptide, respectively.Thick tubes represent disulfide bonds (three in each ICK domain).

FIG. 9. Functional characterisation of individual Hi1a inhibitor cystineknot (ICK) domains. Top: Alignment of the amino acid sequences of thetwo ICK domains in Hi1a. Cysteine residues are highlighted by white texton a black background (Hi1a-N—SEQ ID NO:17; Hi1a-C—SEQ ID NO:36).Bottom: The graph shows concentration-effect curves for inhibition ofrASIC1a by variants of the N-terminal ICK domain (Hi1a-N) and theC-terminal ICK domain (Hi1a-C) (number of experiments=5). I/I_(max):test current/control current.

FIG. 10. Representative recordings from oocytes expressing rASIC1a.Whole-cell currents were elicited by a change in extracellular pH from7.45 to 6.0, every 60 seconds. Oocytes expressing channels were exposedto peptides at pH 7.45 for 60 seconds. Top panel: Hi1a-N applied firstat 3 μM; middle panel: Hi1a-C applied first at 10 μM; bottom panel: BothHi1a-N and Hi1a-C premixed in solution at 3 μM and 10 μM respectivelybefore applications.

FIG. 11. Functional characterisation of Hi1a mutants. Top: Alignment ofthe amino acid sequences of PcTx1 (SEQ ID NO:15) and Hi1a (SEQ IDNO:16). The key pharmacophore residues K8, W24 and R28 in PcTx1 arereplaced with Leu, Tyr and His, respectively, in Hi1a. These residuesare highlighted in white text on a black background. The graph showsconcentration-effect curves for inhibition of rASIC1a by wild-type Hi1a(WT) and the three Hi1a mutants (L8K, Y24W, and H28R). The correspondingIC₅₀ values are shown at right (number of experiments=5). testcurrent/control current.

FIG. 12. Effect of Hi1a-like double ICK peptides on rASIC1a (number ofexperiments=5 for all). Top: Alignment of the amino acid sequences ofHi1a and the engineered Hi1a-like peptides (Hi1a WT—SEQ ID NO:16; EP1—SEQ ID NO:37; EP 2—SEQ ID NO:38; EP 3—SEQ ID NO:39; PcTx1/Hi1a_C—SEQID NO:40). The linker region between the two ICK domains is highlightedwith a grey box. The graph shows concentration-effect curves forinhibition of rASIC1a by wild-type Hi1a (Hi1a WT) and four Hi1a variants(EP 1, EP 2, EP 3, and PcTx1/Hi1a_C); corresponding IC₅₀ values areshown at right. I/I_(max): test current/control current.

FIG. 13. Functional characterisation of recombinant Hi1d. Top: Alignmentof the amino acid sequences of Hi1a (SEQ ID NO:16) and Hi1d (SEQ IDNO:31). Residues that are identical in the two peptides are highlightedin black on a grey background. The graph shows the concentration-effectcurve for inhibition of rASIC1a by Hi1d (mean±SEM; number ofexperiments=6). I/I_(max): test current/control current (mean±SEM;number of experiments=5).

FIG. 14. SHR were given intracerebroventricular (i.c.v.) PcTx1 (1ng/kg), a PcTx1 double mutant that is inactive on ASIC1a (“PcTx1 mutantiso 2”; 1 ng/kg), Hi1a (2 ng/kg) or vehicle (saline) two hours afterstroke. PcTx1 and Hi1a both reduced infarct size in the (A) cortical(peri-infract zone) and (B) striatal (necrotic core) regions of thebrain, but Hi1a was more effective. The inactive PcTx1 mutant had noeffect on infarct size. Data points are mean±SEM (number ofexperiments=7). *P<0.05 versus vehicle (one-way ANOVA).

FIG. 15. Intracerebroventricular (i.c.v.) administration of Hi1a at 2,4, or 8 hours post-stroke reduces infarct size in both the cortical(peri-infract zone) and striatal (necrotic core) regions. (a)Intracerebroventricular (i.c.v.) administration of vehicle (saline) orHi1a (2 ng/kg) at 2, 4, or 8 hours post-stroke. Vehicle: 2 hours, numberof experiments=10; 4 hours, number of experiments=7; 8 hours, number ofexperiments=9. Hi1a: 2 hours, number of experiments=5; 4 hours, numberof experiments=7; 8 hours, number of experiments=10. Data points aremean±SEM. Volumes were measured at 72 hours post-stroke and correctedfor edema. *P<0.05, **P<0.01, ***P<0.001 versus vehicle (one-way ANOVA).(b) Coronal sections showing typical infarcted (darker area) andnon-infarcted regions from spontaneously hypertensive rats (SHR) treatedi.c.v. with either vehicle or Hi1a (2 ng/kg) 8 hours after stroke.

FIG. 16. Reduction in infarct size with Hi1a administration correlateswith improved behavioural scores after stroke. (a) Neurological scoresmeasured pre-stroke (PS) and 24 and 72 hours post-stroke (ps). Datapoints are mean±SEM. ##p<0.01 versus pre-stroke performance; **p<0.01versus corresponding time in vehicle-treated group (two-way repeatedmeasures ANOVA followed by Tukey post hoc tests). (b) Motor score (%error in ledged beam test) measured pre-stroke (PS) and 24 and 72 hourspost-stroke. Data points are mean±SEM. ##p<0.01 versus pre-strokeperformance; *p<0.05, **p<0.01 versus corresponding time invehicle-treated group (two-way repeated measures ANOVA followed by Tukeypost hoc tests).

FIG. 17. The reduction in infarct size with Hi1a administration (FIG.15) correlates with improved neuronal survival. Neuronal survival in thepenumbral (cortical) and core (striatal) regions of damage was measured72 hours post-stroke. Data are expressed as the number (mean±SEM) ofNeuN-immunopositive (NeuN⁺) cells per 0.4 mm² within the non-occluded(contralateral) and occluded (ipsilateral) hemispheres. **p<0.01 versusvehicle-treated group (ipsilateral side); ##p<0.01 versus matched regionon non-infarcted hemisphere (two-way ANOVA followed by Tukey post hoctests).

FIG. 18. Intranasal (i.n.) administration of Hi1a reduces infarct sizein both the cortical (peri-infract zone) and striatal (necrotic core)regions. Compared with i.n. administration of vehicle, i.n.administration of Hi1a at a dose of 2 μg/kg four hours after strokereduces infarct size in both (A) cortical and (B) striatal regions ofthe brain, reduces sensorimotor deficits measured using the ledged beamassay (C), improves overall neurological score (D) and minimises weightloss (E). Infarct volume was measured at 72 hours after stroke.Behavioural tests were carried out pre-stroke (PS) and 24 and 72 hourspost-stroke. Data points are mean±SEM. Vehicle: number of experiments=6.Hi1a (2 μg/kg): number of experiments=7. *P<0.05 vs corresponding timepoint in vehicle, **P<0.001 vs corresponding time point in vehicle.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO:1=nucleic acid coding sequence of Hi1a-precursor (390 nucleicacids).

SEQ ID NO:2=peptide sequence of Hi1a-precursor (129 amino acids).

SEQ ID NO:3=nucleic acid coding sequence of Hi1b-precursor (390 nucleicacids).

SEQ ID NO:4=peptide sequence of Hi1b-precursor (129 amino acids).

SEQ ID NO:5=nucleic acid coding sequence of Hi1c-precursor (456 nucleicacids).

SEQ ID NO:6=peptide sequence of Hi1c-precursor (151 amino acids).

SEQ ID NO:7=nucleic acid coding sequence of Hi1d-precursor (387 nucleicacids).

SEQ ID NO:8=peptide sequence of Hi1d-precursor (128 amino acids).

SEQ ID NO:9=nucleic acid coding sequence of Hi1e_1-precursor (390nucleic acids).

SEQ ID NO:10=peptide sequence of Hi1e_1-precursor (129 amino acids).

SEQ ID NO:11=nucleic acid coding sequence of Hi1e_2-precursor (390nucleic acids).

SEQ ID NO:12=peptide sequence of Hi1e_2-precursor (129 amino acids).

SEQ ID NO:13=nucleic acid coding sequence of Hi1a for expression inEscherichia coli (228 nucleic acids).

SEQ ID NO:14=peptide sequence of recombinant Hi1a expressed in E. coli(76 amino acids including N-terminal serine which is a vestige of theprotease cleavage site).

SEQ ID NO:15=peptide sequence of native mature PcTx1 (40 amino acids).

SEQ ID NO:16=peptide sequence of native mature Hi1a (75 amino acids)

SEQ ID NO:17=peptide sequence of the N-terminal ICK domain of Hi1a (37amino acids).

SEQ ID NO:18=peptide sequence of the N-terminal ICK domain of Hi1a witha modified C-terminus (VP) and N-terminal serine as vestige ofexpression system (36 amino acids).

SEQ ID NO:19=peptide sequence of the N-terminal ICK domain of Hi1a withN-terminal serine as vestige of expression system (38 amino acids)

SEQ ID NO:20=peptide sequence of the N-terminal ICK domain of Hi1a witha modified C-terminus (IPG) and N-terminal serine as vestige ofexpression system (39 amino acids).

SEQ ID NO:21=peptide sequence of the C-terminal ICK domain of Hi1a (40amino acids)

SEQ ID NO:22=peptide sequence of recombinant Hi1a L8K (76 amino acids)

SEQ ID NO:23=peptide sequence of recombinant Hi1a Y24W (76 amino acids)

SEQ ID NO:24=peptide sequence of recombinant Hi1a H28R (76 amino acids).

SEQ ID NO:25=peptide sequence of recombinant engineered peptide #1 (78amino acids)

SEQ ID NO:26=peptide sequence of recombinant engineered peptide #2 (78amino acids)

SEQ ID NO:27=peptide sequence of recombinant engineered peptide #3 (79amino acids)

SEQ ID NO:28=peptide sequence of recombinant engineered peptide #4,PcTx1:Hi1a_C, the C-terminal ICK domain of Hi1a linked to PcTx1 (81amino acids).

SEQ ID NO:29=peptide sequence of native mature Hi1b (75 amino acids).

SEQ ID NO:30=peptide sequence of native mature Hi1c (100 amino acids).

SEQ ID NO:31=peptide sequence of native mature Hi1d (77 amino acids).

SEQ ID NO:32=peptide sequence of native mature Hi1e_1 (75 amino acids).

SEQ ID NO:33=peptide sequence of native mature Hi1e_2 (75 amino acids).

SEQ ID NO:34=nucleic acid sequence of the coding sequence used toproduce recombinant Hi1a (1416 nucleic acids).

SEQ ID NO:35=amino acid sequence of the full precursor protein forrecombinant Hi1a (470 residues).

Description Of Embodiments

The present invention is predicated in part on the discovery of a noveldisulfide-rich peptide, termed Hi1a, from the venom of the Australianfunnel-web spider, Hadronyche infensa. Hi1a is an 8.6-kDa peptidecontaining six disulfide bonds. Hi1a is a double-knot toxin (Bohlen etal., 2010) in which two inhibitor cystine knot (ICK) motifs are joinedhead-to-tail by a six-residue linker. The ICK motif is a common featureof spider-venom peptides, and it typically results in these peptideshaving high levels of chemical and thermal stability as well asresistance to proteases. The present inventors have found that Hi1a isthe most potent inhibitor of human ASIC1a described to date, and thatthe peptide is highly selective for ASIC1a over other ASIC subtypes.

By analysing a venom-gland transcriptome from the Australian funnel-webspider, Hadronyche infensa, the present inventors identified Hi1a and anumber of paralogous peptides.

In a first aspect, the present invention provides an isolated, syntheticor recombinant disulfide-rich peptide with neuroprotective activity. Thepeptide comprises, consists of, or consists essentially of, a sequenceof Formula (I):

X-L-Y   (I)

wherein X and Y each represent a peptide sequence having an inhibitorcystine knot (ICK) fold and L is a linker, and wherein said peptide ispreferably capable of specifically binding to ASIC1a.

For the purposes of this invention, “isolated” means material that hasbeen removed from its natural state or otherwise subjected to humanmanipulation. Isolated material may be substantially or essentially freefrom components that normally accompany it in its natural state, or maybe manipulated so as to be in an artificial state. Isolated materialcould be material in native or recombinant form. The term “isolated”also encompasses terms such as “purified” and/or “synthetic”.

The term “recombinant” as used herein with regards to a peptide orprotein means a peptide or protein produced by recombinant techniques.

By “disulfide-rich peptide” in the context of this invention, is meant apeptide having six cysteine residues which are bonded in pairs to formthree disulfide bonds. Preferably, the three disulfide bonds form apseudo-knot known as an inhibitor cystine knot (ICK) motif. Particularlypreferred disulfide-rich peptides of the invention comprise twelvecysteine residues covalently joined in pairs to form six disulfidebonds, such that the peptide comprises two ICK motifs.

As used herein, “neuroprotective activity” refers to the effect ofreducing or ameliorating damage to neuronal and/or glial cells or tissuesuch as can occur during stroke. As such, “neuroprotective activity”results in reduced severity of symptoms relating to, for example,sensorimotor function and cognitive ability.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of”. Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they affect theactivity or action of the listed elements.

In the sequence of Formula (I), X and Y can be different sequences orthey can be the same sequence. In preferred embodiments X and Y aredifferent sequences, but both fold to form an ICK motif. Each X and Yis, therefore, preferably a sequence of about 25 to about 50 aminoacids, even more preferably, about 30, 31, 32, 33, 34, 35, 36, 37, 38,39 or 40 amino acids.

In particular embodiments, X has the sequence defined by residues 55 to87 of SEQ ID NO:2, residues 52 to 84 of SEQ ID NO:6, or residues 1 to 34of SEQ ID NO:14. Particularly preferred embodiments are those where Xhas the sequence defined by residues 55 to 87 of SEQ ID NO:2, orresidues 1 to 34 of SEQ ID NO:14.

Independently of X, in particular embodiments, Y has the sequencedefined by residues 94 to 129 of SEQ ID NO:2, residues 94 to 129 of SEQID NO:4, residues 101 to 151 of SEQ ID NO:6, residues 93 to 128 of SEQID NO:8, or residues 94 to 129 of SEQ ID NO:10. In a particularlypreferred embodiment, Y has the sequence defined by residues 94 to 129of SEQ ID NO:2.

The linker, L, can be a bond, a non-amino acid-based chemical moiety, asingle amino acid, or a peptide sequence. For embodiments where L is asingle amino acid, L can be a naturally occurring amino acid or asynthetic, non-naturally occurring amino acid such as a chemicalanalogue of a corresponding naturally occurring amino acid. In preferredembodiments, L is a peptide sequence of at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or 16 amino acids. The amino acids of thesequence can be the same natural or non-natural amino acid, or eachamino acid of the sequence can be independently selected from anaturally occurring amino acid or a synthetic, non-naturally occurringamino acid such as a chemical analogue of a corresponding naturallyoccurring amino acid. In a particularly preferred embodiment, L is apeptide sequence of six naturally occurring amino acids.

In certain embodiments, L comprises predominantly (that is, more than50% of the amino acid residues of the linker) naturally occurringhydrophobic amino acids Amino acids having a hydrophobic side chaininclude tyrosine, valine, isoleucine, leucine, methionine,phenylalanine, tryptophan, alanine and proline.

In particular embodiments, the linker can have the sequence defined byresidues 88 to 93 of SEQ ID NO:2, residues 85 to 100 of SEQ ID NO:6, orresidues 85 to 92 of SEQ ID NO:8. In a particularly preferredembodiment, the linker has the sequence defined by residues 88 to 93 ofSEQ ID NO:2.

The peptides of Formula (I) can comprise any combination of X, Y and Lto provide a disulfide-rich peptide capable of specifically binding toASIC1a. Particularly preferred embodiments comprise a peptide wherein Xhas the sequence defined by residues 55 to 87 of SEQ ID NO:2 or residues1 to 34 of SEQ ID NO:14, L has the sequence defined by residues 88 to 93of SEQ ID NO:2, and Y has the sequence defined by residues 94 to 129 ofSEQ ID NO:2, wherein said peptide is capable of specifically binding toASIC1a.

Preferably, the peptides of Formula (I) have a half-maximal inhibitoryconcentration (IC₅₀) for inhibiting ASIC1a of less than about 10 nM.Even more preferably, the peptides inhibit ASIC1a with an IC₅₀ of lessthan about 1 nM.

In a second aspect, the present invention provides a functionally activeneuroprotective fragment, derivative or analogue of the disulfide-richpeptide provided by the first aspect that is preferably capable ofspecifically binding to ASIC1a.

As used herein, the term “functionally active” means retaining at least50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% of theneuroprotective activity of the disulfide-rich peptide provided by thefirst aspect.

The term “fragment” as used herein refers to a sequence that constitutesless than 100%, but at least 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 96, 97, 98 or 99% of the disulfide-rich peptide.

A “derivative” of the disulfide-rich peptide is a peptide that has beenaltered, for example by conjugation or complexing with a chemicalmoiety, by post-translational modification (including, but not limitedto, phosphorylation, glycosylation, acetylation, lipidation orpegylation), or by the addition of one or more amino acids (including,for example, the addition of a protein or tag to assist withpurification). The incorporation of non-naturally occurring amino acidsis also encompassed by “derivative”.

As used herein, the term “analogue” refers to a compound that isstructurally similar to the disulfide-rich peptide provided by the firstaspect, but differs in some aspect.

Preferably, the functionally active neuroprotective fragment, derivativeor analogue has a half-maximal inhibitory concentration (IC₅₀) forinhibiting ASIC1a of less than about 10 nM. Even more preferably, thefunctionally active neuroprotective fragment, derivative or analogueinhibits ASIC1a with an IC₅₀ of less than about 1 nM.

In a third aspect, the present invention provides a neuroprotectivepeptide in which two ICK motifs are joined head-to-tail by a six-residuelinker, and wherein said peptide is preferably capable of specificallybinding to ASIC1a.

The six residues of the linker can be the same naturally occurring ornon-naturally occurring amino acid, or each residue of the linker can beindependently selected from naturally occurring and non-naturallyoccurring amino acids. In a particularly preferred embodiment, L is apeptide sequence of six naturally occurring amino acids.

In certain embodiments, the linker comprises predominantly hydrophobicamino acid residues, that is, 4, 5 or 6, naturally occurring hydrophobicamino acids. In a particularly preferred embodiment, the linker has thesequence defined by residues 88 to 93 of SEQ ID NO:2.

The two ICK motifs can be different sequences or they can be the samesequence. In preferred embodiments, the two ICK motifs have differentsequences. Each ICK motif is preferably a sequence of about 25 to about50 amino acids, even more preferably, about 30, 31, 32, 33, 34, 35, 36,37, 38, 39 or 40 amino acids.

In particular embodiments, the N-terminal ICK motif has the sequencedefined by residues 55 to 87 of SEQ ID NO:2, residues 52 to 84 of SEQ IDNO:6, or residues 1 to 34 of SEQ ID NO:14. Particularly preferredembodiments are those where the N-terminal ICK motif has the sequencedefined by residues 55 to 87 of SEQ ID NO:2, or residues 1 to 34 of SEQID NO:14.

Independently of the N-terminal ICK motif, in particular embodiments,the C-terminal ICK motif has the sequence defined by residues 94 to 129of SEQ ID NO:2, residues 94 to 129 of SEQ ID NO:4, residues 101 to 151of SEQ ID NO:6, residues 93 to 128 of SEQ ID NO:8, or residues 94 to 129of SEQ ID NO:10. In a particularly preferred embodiment, the C-terminalICK motif has the sequence defined by residues 94 to 129 of SEQ ID NO:2.

The peptides can comprise any combination of ICK motifs with asix-residue linker to provide a neuroprotective peptide capable ofspecifically binding to ASIC1a. Particularly preferred embodimentscomprise a peptide wherein the N-terminal ICK motif has the sequencedefined by residues 55 to 87 of SEQ ID NO:2 or residues 1 to 34 of SEQID NO:14, the linker has the sequence defined by residues 88 to 93 ofSEQ ID NO:2, and the C-terminal ICK motif has the sequence defined byresidues 94 to 129 of SEQ ID NO:2, wherein said peptide is capable ofspecifically binding to ASIC1a.

Preferably, the neuroprotective peptides have a half maximal inhibitoryconcentration (IC₅₀) for inhibiting ASIC1a of less than about 10 nM.Even more preferably, the peptides inhibit ASIC1a with an IC₅₀ of lessthan about 1 nM.

In a fourth aspect, the present invention provides a neuroprotectivepeptide comprising twelve cysteine residues covalently joined in pairsto form six disulfide bonds, such that the peptide comprises two ICKmotifs, and wherein said peptide is preferably capable of specificallybinding to ASIC1a. Preferably, the two ICK motifs are joinedhead-to-tail such that the peptide comprises two concatenated ICKmotifs. The two ICK motifs can be joined directly (that is, via apeptide bond) or they can be joined by a linker. The linker can be anon-amino acid-based chemical moiety, a single amino acid, or a peptidesequence. For embodiments where the linker is a single amino acid, thelinker can be a naturally occurring or a non-naturally occurring aminoacid. For embodiments where the linker is a peptide sequence, thepeptide sequence can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16 amino acids. The amino acids of the linker can be the samenatural or non-natural amino acid, or each amino acid of the linkersequence can be independently selected from naturally occurring andnon-naturally occurring amino acids. In a particularly preferredembodiment, the linker is a peptide sequence of six naturally occurringamino acids.

In certain embodiments, the linker comprises predominantly (that is,more than 50% of the amino acid residues of the linker) naturallyoccurring hydrophobic amino acids. In particular embodiments, the linkercan have the sequence defined by residues 88 to 93 of SEQ ID NO:2,residues 85 to 100 of SEQ ID NO:6, or residues 85 to 92 of SEQ ID NO:8.In a particularly preferred embodiment, the linker has the sequencedefined by residues 88 to 93 of SEQ ID NO:2.

The two ICK motifs can be different sequences or they can be the samesequence. In preferred embodiments, the two ICK motifs have differentsequences. Each ICK motif is preferably a sequence of about 25 to about50 amino acids, even more preferably, about 30, 31, 32, 33, 34, 35, 36,37, 38, 39 or 40 amino acids.

In particular embodiments, the N-terminal ICK motif has the sequencedefined by residues 55 to 87 of SEQ ID NO:2, residues 52 to 84 of SEQ IDNO:6, or residues 1 to 34 of SEQ ID NO:14. Particularly preferredembodiments are those where the N-terminal ICK motif has the sequencedefined by residues 55 to 87 of SEQ ID NO:2, or residues 1 to 34 of SEQID NO:14.

Independently of the N-terminal ICK motif, in particular embodiments,the C-terminal ICK motif has the sequence defined by residues 94 to 129of SEQ ID NO:2, residues 94 to 129 of SEQ ID NO:4, residues 101 to 151of SEQ ID NO:6, residues 93 to 128 of SEQ ID NO:8, or residues 94 to 129of SEQ ID NO:10. In a particularly preferred embodiment, the C-terminalICK motif has the sequence defined by residues 94 to 129 of SEQ ID NO:2.

The peptides can comprise any combination of ICK motifs to provide aneuroprotective peptide capable of specifically binding to ASIC1a.Particularly preferred embodiments comprise a peptide wherein theN-terminal ICK motif has the sequence defined by residues 55 to 87 ofSEQ ID NO:2 or residues 1 to 34 of SEQ ID NO:14, and the C-terminal ICKmotif has the sequence defined by residues 94 to 129 of SEQ ID NO:2,wherein said peptide is capable of specifically binding to ASIC1a.

Preferably, the peptides have a half-maximal inhibitory concentration(IC₅₀) for inhibiting ASIC1a of less than about 10 nM. Even morepreferably, the peptides inhibit ASIC1a with an IC₅₀ of less than about1 nM.

In a fifth aspect, the present invention provides an isolated, syntheticor recombinant disulfide-rich peptide with neuroprotective activity. Thepeptide comprises, consists of, or consists essentially of, an aminoacid sequence selected from the group consisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8,        10, 12, 14 or 16;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 or 16;    -   (c) a sequence that is encoded by the nucleotide sequence set        forth in any one of SEQ ID NOs:1, 3, 5, 7, 9, 11 or 13;    -   (d) a sequence that is encoded by a nucleotide sequence that        shares at least 65% (and at least 66% to at least 99% and all        integer percentages in between) sequence similarity or sequence        identity with the sequence set forth in any one of SEQ ID NOs:        1, 3, 5, 7, 9, 11 or 13; or    -   (e) a sequence that is encoded by a nucleotide sequence that        hybridizes under at least medium or high stringency conditions        to the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13.

The phrase “and all integer percentages in between”, as used hereinrefers to increasing the lower of the two stated values by the integer‘1’ until the higher of the two stated values is reached. For example,“and at least 66% to at least 99% and all integer percentages inbetween” refers to at least 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%.

“Sequence similarity” refers to the percentage number of amino ornucleic acids that are identical or constitute conservativesubstitutions.

The term “sequence identity” as used herein, refers to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base or theidentical amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity.

“Hybridizes” is used herein to refer to the pairing of at least partlycomplementary nucleotide sequences to produce a DNA-DNA, RNA-RNA orDNA-RNA hybrid. Hybrid sequences comprising complementary nucleotidesequences occur through base-pairing.

“Medium or high stringency conditions” refers to the temperature andionic strength conditions under which sequences will hybridize. Thehigher the stringency, the higher will be the required level ofcomplementarity between hybridizing nucleotide sequences. Stringencyconditions are well-known in the art, with a skilled addressee able torecognize that various factors can be manipulated to optimize thespecificity of hybridization (see, for example, Green and Sambrook,2012).

In a preferred embodiment, the isolated, synthetic or recombinantdisulfide-rich peptide comprises SEQ ID NO:16. In a particularlypreferred embodiment, the isolated, synthetic or recombinantdisulfide-rich peptide consists of SEQ ID NO:14.

In a sixth aspect, the present invention provides an isolated, syntheticor recombinant disulfide-rich peptide derived from spider venom that iscapable of specifically binding to ASIC1a. In particular embodiments,the peptide can be derived from the venom of any Australian funnel-webspider from within the genera Hadronyche, Atrax or Illawarra, or arelated mygalomorph spider.

As used herein, “derived” in one sense simply means that the peptide wasoriginally identified in the spider venom. In another sense, “derived”refers to a disulfide-rich peptide that differs from a native peptide byvirtue of deletion (truncation) or addition of one or more amino acidsto the N-terminal and/or C-terminal end of the native protein, deletionor addition of one or more amino acids at one or more positions withinthe sequence of the native protein, or substitution of one or more aminoacids at one or more sites within the sequence of the native protein.

In a seventh aspect, the present invention provides an isolated,synthetic or recombinant nucleic acid molecule that comprises, consistsof, or consists essentially of, a nucleotide sequence encoding the aminoacid sequence of the disulfide-rich peptide provided by the first orsixth aspects, the fragment, derivative or analogue provided by thesecond aspect, or the neuroprotective peptide provided by the third orfourth aspects. In some embodiments, the nucleic acid moleculecomprises, consists of, or consists essentially of, a nucleotidesequence selected from the group consisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 or 13, or a complement        thereof; or    -   (c) a sequence that hybridizes under at least medium or high        stringency conditions to the sequence set forth in any one of        SEQ ID NOs:1, 3, 5, 7, 9, 11 or 11, or a complement thereof.

In a preferred embodiment, the isolated nucleic acid molecule comprisesSEQ ID NO:13. In a particularly preferred embodiment, the isolatednucleic acid molecule consists of SEQ ID NO:13.

In an eighth aspect, the present invention provides a genetic constructfor expressing the nucleic acid molecule provided by the seventh aspect.The genetic construct comprises the isolated nucleic acid moleculeprovided by the seventh aspect operably linked to one or more regulatorysequences in an expression vector.

Typically, the genetic construct is in the form of, or comprises geneticcomponents of, a plasmid, bacteriophage, a cosmid, a yeast or bacterialartificial chromosome as are well understood in the art (Green andSambrook, 2012). Genetic constructs may be suitable for maintenance andpropagation of the isolated nucleic acid in bacteria or other hostcells, for manipulation by recombinant DNA technology and/or expressionof the nucleic acid of the second aspect or an encoded protein of theinvention.

For the purposes of host cell expression, the genetic construct is anexpression construct. Typically, the expression construct comprises thenucleic acid molecule provided by the seventh aspect operably linked toone or more additional sequences in an expression vector. An “expressionvector” can be either a self-replicating extra-chromosomal vector suchas a plasmid, or a vector that integrates into a host genome (Green andSambrook, 2012).

The phrase “operably linked” as used herein means placing additionalnucleotide sequence(s) relative to the nucleic acid of the second aspectwithin the genetic construct, wherein the additional nucleotidesequence(s) initiates, regulates or otherwise controls transcription.

Regulatory nucleotide sequences are selected as being appropriate forthe host cell used for expression. Numerous types of appropriateexpression vectors and suitable regulatory sequences for a variety ofhost cells are known in the art. The regulatory sequences can include,but are not limited to, promoter sequences (constitutive or inducible),leader or signal sequences, ribosomal binding sites, transcriptionalstart and termination sequences, translational start and terminationsequences, and enhancer or activator sequences.

In some embodiments, the genetic construct further comprises one or morenucleotide sequences such that the disulfide-rich peptide of theinvention is expressed as a fusion protein. Preferably the fusionprotein comprises a disulfide-rich peptide of the first aspect and oneor more moieties. The one or more moieties are known in the art asprotein tags. Protein tags are selected based on function and includeaffinity tags, such as maltose binding protein (MBP), chitin bindingprotein (CBP), glutathione S-transferase (GST) and poly(His) tag(usually a hexa-histidine tag), and solubilisation tags, such as MBP andGST. The presence of an affinity tag can assist with purification of thefusion protein. For example, a fusion protein comprising a poly(His) tagcan be readily purified using immobilized metal ion affinitychromatography (IMAC). The presence of a solubilisation tag can assistwith solubility and folding of the expressed protein. Tags such as MBPand GST have dual functionality as affinity tags and solubilisation tags(Sachev and Chirgwin, 2000; Smith, 2000).

In a preferred embodiment of the invention, the genetic constructcomprises nucleotide sequences such that the disulfide-rich peptide ofthe invention is expressed as a fusion protein comprising an affinitytag and a solubilisation tag. In a particularly preferred embodiment,the affinity tag is a poly(His) tag and the solubilisation tag is MBP.The fusion protein preferably further comprises a cleavage site for easeof release of the disulfide-rich peptide from the tags.

In a ninth aspect, the present invention provides a host celltransformed with the nucleic acid molecule provided by the seventhaspect or the genetic construct provided by the eighth aspect.

The host cell can be prokaryotic or eukaryotic. For example, suitablehost cells include mammalian cells (such as HeLa, HEK293T or Jurkat),yeast cells (such as Pichia pastoris), insect cells (such as Sf9 orTrichoplusia ni) utilized with or without a baculovirus expressionsystem, or bacterial cells (such as E. coli) (Klint et al., 2013).

In a particularly preferred embodiment, the host cell is E. coli.

In an even more preferred embodiment, the host cell is E. coli and thedisulfide-rich peptide is expressed in the periplasm to take advantageof the disulfide-bond machinery (Dsb system) located in this region ofthe cell (Klint et al., 2013).

In an alternative embodiment, the disulfide-rich peptides of theinvention are produced in a cell-free expression system. Cell-freeprotein synthesis (also referred to as in vitro translation) has beensuccessfully used for the production of proteins, includingdisulfide-bonded proteins (Kim and Swartz, 2004; Bundy and Swartz,2011).

In a tenth aspect, the present invention provides a method of producingthe isolated, synthetic or recombinant disulfide-rich peptide providedby the first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects, comprising culturing the transformedhost cell provided by the ninth aspect and isolating the resultantpeptide, fragment, derivative or analogue from said cultured host cell.

The method can comprise further steps, including purification and/orfolding. For example, in embodiments where the disulfide-rich peptide isexpressed as a fusion protein comprising an affinity tag, as describedherein, the method can further comprise purification of the fusionprotein using a process suited to the affinity tag. In the case of apoly(His) tag, the purification step can comprise IMAC.

Expression of disulfide-rich peptides does not always result information of the correct disulfide bonds, and therefore a non-nativefold (three-dimensional structure) can result. A further step in theproduction of the isolated disulfide-rich peptide of the first aspectcan therefore comprise the use of a redox buffer to assist in correctdisulfide-bond formation. To avoid interference from impurities,preferably, a redox buffer is utilised after the expressed protein hasbeen purified from the cell culture.

The method can further comprise cleavage of the fusion protein from anyaffinity and/or solubilisation tags.

In an eleventh aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of at leastone isolated, synthetic or recombinant disulfide-rich peptide providedby the first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects.

The phrase “pharmaceutical composition” as used herein encompassespharmaceutical and veterinary compositions.

Pharmaceutically acceptable carriers, diluents and excipients which canbe used in the pharmaceutical compositions of the invention will beknown to those of skill in the art. The British Pharmacopoeia (BP) andthe United States Pharmacopeia and National Formulary (USP-NF) containdetails of suitable carriers, diluents and excipients, as does SweetmanS (Ed.), ‘Martindale: The complete drug reference.’ London:Pharmaceutical Press, 37th Ed., (2011), and Rowe R C, Sheskey P J, QuinnM E (Ed.), ‘Handbook of Pharmaceutical Excipients’, 6th Ed., London:Pharmaceutical Press (2009), the contents of which are incorporatedherein by cross reference.

The pharmaceutical composition can be formulated for administration byany suitable route. In certain embodiments, the composition is suitablefor parenteral administration. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. A particularlypreferred mode of administration is intranasal (i.n.).

For i.c.v. or intrathecal administration, the pharmaceutical compositioncan be formulated in a carrier, such as a diluent, adjuvant, excipient,buffer, stabiliser, isotonicising agent, preservative or anti-oxidant.Preferably, the composition is formulated in a parenterally acceptableaqueous solution which has suitable pH, isotonicity and stability.

For i.n. administration, the peptide, fragment, derivative or analoguecan be formulated in a liquid carrier suitable for delivery as a nasalspray. The composition may additionally comprise a delivery enhancer toassist with transport of the peptide, fragment, derivative or analogueacross the nasal mucosa. The delivery enhancer can comprise, forexample, lipids, polymers, liposomes, emulsions, or nanoparticles.

Similarly, for buccal delivery, the peptide, fragment, derivative oranalogue can be formulated in a suitable liquid carrier. The compositionpreferably further comprises a delivery enhancer to assist withtransport of the peptide, fragment, derivative or analogue across thebuccal mucosa. The delivery enhancer can comprise, for example, lipids,polymers, liposomes, emulsions, or nanoparticles.

In a twelfth aspect, the invention provides a method for the treatmentof stroke in a subject comprising the step of administering atherapeutically effective amount of at least one isolated, synthetic orrecombinant disulfide-rich peptide provided by the first or sixthaspects, the fragment, derivative or analogue provided by the secondaspect, the neuroprotective peptide provided by the third or fourthaspects or the pharmaceutical composition provided by the eleventhaspect.

The subject for treatment can be a human, mammal or animal. Preferably,the subject is a human or other type of mammal.

A “therapeutically effective amount” is the amount effective fortreating or lessening the severity of one or more symptoms associatedwith stroke, including neurological function, neuronal damage,sensorimotor function and cognitive ability.

In preferred embodiments, the therapeutically effective amount of thepeptide, fragment, derivative, analogue or pharmaceutical composition is1-10 ng/kg for i.c.v. delivery and 1-10 μg/kg for intranasal delivery.

Preferably, the step of administering a therapeutically effective amountof at least one isolated, synthetic or recombinant disulfide-richpeptide provided by the first or sixth aspects, the fragment, derivativeor analogue provided by the second aspect, the neuroprotective peptideprovided by the third or fourth aspects or the pharmaceuticalcomposition provided by the eleventh aspect is undertaken as soon aspossible after the onset of stroke. The step of administering ispreferably undertaken at any time from the onset of the stroke to about12 hours after the onset of stroke. The step of administering istherefore undertaken no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or1 hour(s) after the onset of stroke. Preferably, the step ofadministering is undertaken no more than 8 hours after the onset ofstroke. In an alternative preferred embodiment, the step ofadministering is undertaken no more than 4 hours after the onset ofstroke.

The peptide, fragment, derivative, analogue, or pharmaceuticalcomposition is preferably administered via the i.c.v. or i.n. route.

In a thirteenth aspect, the invention provides a method for theprevention or treatment of neuronal damage following stroke in a subjectcomprising the step of administering a therapeutically effective amountof at least one isolated, synthetic or recombinant disulfide-richpeptide provided by the first or sixth aspects, the fragment, derivativeor analogue provided by the second aspect, the neuroprotective peptideprovided by the third or fourth aspects or the pharmaceuticalcomposition provided by the eleventh aspect.

In a fourteenth aspect, the invention provides a method for reducinginfarct size following stroke in a subject comprising the step ofadministering a therapeutically effective amount of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, the neuroprotective peptide provided bythe third or fourth aspects or the pharmaceutical composition providedby the eleventh aspect.

The subject for treatment can be a human, mammal or animal. Preferably,the subject is a human or other type of mammal.

In preferred embodiments, the therapeutically effective amount of thepeptide, fragment, derivative, analogue or pharmaceutical composition is1-10 ng/kg for i.c.v. delivery and 1-10 μg/kg for intranasal delivery.

The step of administering is preferably undertaken at any time from theonset of the stroke to about 12 hours after the onset of stroke. Thestep of administering is therefore undertaken no more than 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 hour(s) after the onset of stroke.Preferably, the step of administering is undertaken no more than 8 hoursafter the onset of stroke. In an alternative preferred embodiment, thestep of administering is undertaken no more than 4 hours after the onsetof stroke.

The peptide, fragment, derivative, analogue, or pharmaceuticalcomposition is preferably administered via the i.c.v. or i.n. route.

In a fifteenth aspect, the invention provides a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the first or sixth aspects, thefragment, derivative or analogue provided by the second aspect, theneuroprotective peptide provided by the third or fourth aspects or thepharmaceutical composition provided by the eleventh aspect for thetreatment of stroke.

In a sixteenth aspect, the invention provides a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the first or sixth aspects, thefragment, derivative or analogue provided by the second aspect, theneuroprotective peptide provided by the third or fourth aspects or thepharmaceutical composition provided by the eleventh aspect for theprevention or treatment of neuronal damage following stroke in asubject.

In a seventeenth aspect, the invention provides use of a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the first or sixth aspects, thefragment, derivative or analogue provided by the second aspect, theneuroprotective peptide provided by the third or fourth aspects or thepharmaceutical composition provided by the eleventh aspect for thereduction of infarct size following stroke in a subject.

The subject for treatment can be a human, mammal or animal. Preferably,the subject is a human or other type of mammal.

In preferred embodiments, the therapeutically effective amount of thepeptide, fragment, derivative, analogue or pharmaceutical composition is1-10 ng/kg for i.c.v. delivery and 1-10 μg/kg for intranasal delivery.

The step of administering is preferably undertaken at any time from theonset of the stroke to about 12 hours after the onset of stroke. Thestep of administering is therefore undertaken no more than 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 hour(s) after the onset of stroke.Preferably, the step of administering is undertaken no more than 8 hoursafter the onset of stroke. In an alternative preferred embodiment, thestep of administering is undertaken no more than 4 hours after the onsetof stroke.

In an eighteenth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects in the manufacture of a medicament forthe treatment of stroke.

The medicament can be formulated for parenteral administration.Preferably, the medicament is formulated for administration via thei.c.v. or i.n. route.

In preferred embodiments, the medicament is formulated to providebetween 1 ng/kg and 2 μg/kg of the peptide, fragment, derivative oranalogue. In particularly preferred embodiments, the medicament isformulated to provide 1-2 ng/kg for i.c.v. delivery and 1-2 μg/kg forintranasal delivery.

In a nineteenth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects in the manufacture of a medicament forthe prevention or treatment of neuronal damage following stroke in asubject.

The medicament can be formulated for parenteral administration.Preferably, the medicament is formulated for administration via thei.c.v. or i.n. route.

In preferred embodiments, the medicament is formulated to providebetween 1 ng/kg and 2 μg/kg of the peptide, fragment, derivative oranalogue. In particularly preferred embodiments, the medicament isformulated to provide 1-2 ng/kg for i.c.v. delivery and 1-2 μg/kg forintranasal delivery.

In a twentieth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe first or sixth aspects, the fragment, derivative or analogueprovided by the second aspect, or the neuroprotective peptide providedby the third or fourth aspects in the manufacture of a medicament forthe reduction of infarct size following stroke in a subject.

The medicament can be formulated for parenteral administration.Preferably, the medicament is formulated for administration via thei.c.v. or i.n. route.

In preferred embodiments, the medicament is formulated to providebetween 1 ng/kg and 2 μg/kg of the peptide, fragment, derivative oranalogue. In particularly preferred embodiments, the medicament isformulated to provide 1-2 ng/kg for i.c.v. delivery and 1-2 μg/kg forintranasal delivery.

In a twenty-first aspect, the invention provides use of an isolated,synthetic or recombinant disulfide-rich neuroprotective peptide derivedfrom spider venom for treating stroke. In particular embodiments, thepeptide can be derived from the venom of any Australian funnel-webspider from within the genera Hadronyche, Atrax and Illawarra, or arelated mygalomorph spider.

In a twenty-second aspect, there is provided a method for identifying ordesigning a peptide, peptidomimetic, or small molecule capable ofinhibiting activation of ASIC1a, said method comprising the steps of:

-   -   (i) computer modelling the interaction between ASIC1a and at        least one disulfide-rich peptide, wherein said disulfide-rich        peptide is as defined in any one of the first to sixth aspects;    -   (ii) using data generated by the computer modelling to identify        or design a peptide, peptidomimetic, or small molecule capable        of binding to ASIC1a and inhibiting the activation of ASIC1a;        and optionally,    -   (iii) producing the peptide, peptidomimetic, or small molecule        of step (ii), and optionally,    -   (iv) testing the peptide, peptidomimetic, or small molecule of        step (iii) for binding to ASIC1a and inhibiting the activation        of ASIC1a.

Preferably, the peptide, peptidomimetic, or small molecule is capable ofspecifically binding to ASIC1a. Even more preferably, the peptide,peptidomimetic, or small molecule has an IC₅₀ for inhibiting ASIC1a ofless than about 10 nM. In particularly preferred embodiments, thepeptide, peptidomimetic, or small molecule inhibits ASIC1a with an IC₅₀of less than about 1 nM.

In further preferred embodiments, the peptide, peptidomimetic, or smallmolecule is a peptide falling within the scope of the fifth aspect.

Other particularly preferred aspects of the invention are defined belowand can have one or more features as described in respect of the firstto twenty-second aspects, or as described elsewhere in thisspecification.

In a twenty-third aspect, the present invention provides an isolated,synthetic or recombinant disulfide-rich peptide. The peptide comprises,consists of, or consists essentially of, a sequence of Formula (I):

X-L-Y   (I)

wherein X and Y each represent a peptide sequence having an inhibitorcystine knot (ICK) fold and L is a linker, and wherein said peptide ispreferably capable of specifically binding to acid sensing ion channelsubtype 1a (ASIC1a).

In a twenty-fourth aspect, the present invention provides a functionallyactive fragment, derivative or analogue of the disulfide-rich peptideprovided by the twenty-third aspect that is preferably capable ofspecifically binding to ASIC1a.

In a twenty-fifth aspect, the present invention provides a peptide inwhich two ICK motifs are joined head-to-tail by a six-residue linker,and wherein said peptide is preferably capable of specifically bindingto ASIC1a.

In a twenty-sixth aspect, the present invention provides a peptidecomprising twelve cysteine residues covalently joined in pairs to formsix disulfide bonds, such that the peptide comprises two ICK motifs, andwherein said peptide is preferably capable of specifically binding toASIC1a.

In a twenty-seventh aspect, the present invention provides an isolated,synthetic or recombinant disulfide-rich peptide with therapeutic orprophylactic activity, wherein the peptide comprises, consists of, orconsists essentially of, an amino acid sequence selected from the groupconsisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8,        10, 12, 14 or 16;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14 or 16;    -   (c) a sequence that is encoded by the nucleotide sequence set        forth in any one of SEQ ID NOs:1, 3, 5, 7, 9, 11 or 13;    -   (d) a sequence that is encoded by a nucleotide sequence that        shares at least 65% (and at least 66% to at least 99% and all        integer percentages in between) sequence similarity or sequence        identity with the sequence set forth in any one of SEQ ID NOs:        1, 3, 5, 7, 9, 11 or 13; or    -   (e) a sequence that is encoded by a nucleotide sequence that        hybridizes under at least medium or high stringency conditions        to the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13.

In a twenty-eighth aspect, the present invention provides an isolated,synthetic or recombinant disulfide-rich peptide derived from spidervenom that is capable of specifically binding to ASIC1a and inhibitingan ASIC1a biological pathway.

In a twenty-ninth aspect, the present invention provides an isolated,synthetic or recombinant nucleic acid molecule that comprises, consistsof, or consists essentially of, a nucleotide sequence encoding the aminoacid sequence of the disulfide-rich peptide provided by the twenty-thirdor twenty-eighth aspects, the fragment, derivative or analogue providedby the twenty-fourth aspect, or the peptide provided by the twenty-fifthor twenty-sixth aspects. In some embodiments, the nucleic acid moleculecomprises, consists of, or consists essentially of, a nucleotidesequence selected from the group consisting of:

-   -   (a) the sequence set forth in any one of SEQ ID NOs:1, 3, 5, 7,        9, 11 or 13;    -   (b) a sequence that shares at least 65% (and at least 66% to at        least 99% and all integer percentages in between) sequence        similarity or sequence identity with the sequence set forth in        any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11 or 13, or a complement        thereof; or    -   (c) a sequence that hybridizes under at least medium or high        stringency conditions to the sequence set forth in any one of        SEQ ID NOs:1, 3, 5, 7, 9, 11 or 11, or a complement thereof.

In a thirtieth aspect, the present invention provides a geneticconstruct for expressing the nucleic acid molecule provided by thetwenty-ninth aspect (for example, for making recombinant peptides incommercial quantities). The genetic construct generally comprises theisolated nucleic acid molecule provided by the twenty-ninth aspectoperably linked to one or more regulatory sequences in an expressionvector.

In a thirty-first aspect, the present invention provides a host celltransformed with the nucleic acid molecule provided by the twenty-ninthaspect or the genetic construct provided by the thirtieth aspect.

In a thirty-second aspect, the present invention provides a method ofproducing the isolated, synthetic or recombinant disulfide-rich peptideprovided by the twenty-third or twenty-eighth aspects, the fragment,derivative or analogue provided by the twenty-fourth aspect, or thepeptide provided by the twenty-fifth or twenty-sixth aspects, comprisingculturing the transformed host cell provided by the thirty-first aspectand isolating the resultant peptide, fragment, derivative or analoguefrom said cultured host cell.

In a thirty-third aspect, the present invention provides apharmaceutical composition comprising a therapeutically effective amountof at least one isolated, synthetic or recombinant disulfide-richpeptide provided by the twenty-third or twenty-eighth aspects, thefragment, derivative or analogue provided by the twenty-fourth aspect,or the peptide provided by the twenty-fifth or twenty-sixth aspects.

In a thirty-fourth aspect, the invention provides a method for theprevention or treatment of a condition in a subject caused by ASIC1aactivity or contributed to by ASIC1a activity comprising the step ofadministering a therapeutically effective amount of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe twenty-third or twenty-eighth aspects, the fragment, derivative oranalogue provided by the twenty-fourth aspect, the peptide provided bythe twenty-fifth or twenty-sixth aspects or the pharmaceuticalcomposition provided by the thirty-third aspect.

In a thirty-fifth aspect, the invention provides a therapeuticallyeffective amount of at least one isolated, synthetic or recombinantdisulfide-rich peptide provided by the twenty-third or twenty-eighthaspects, the fragment, derivative or analogue provided by thetwenty-fourth aspect, the peptide provided by the twenty-fifth ortwenty-sixth aspects or the pharmaceutical composition provided by thethirty-third aspect for the prevention or treatment of a condition in asubject caused by ASIC1a activity or contributed to by ASIC1a activity.

In a thirty-sixth aspect, the invention provides use of at least oneisolated, synthetic or recombinant disulfide-rich peptide provided bythe twenty-first or twenty-eighth aspects, the fragment, derivative oranalogue provided by the twenty-fourth aspect, or the peptide providedby the twenty-fifth or twenty-sixth aspects in the manufacture of amedicament for the prevention or treatment of a condition in a subjectcaused by ASIC1a activity or contributed to by ASIC1a activity.

Conditions in subjects caused by ASIC1a activity or contributed to byASIC1a activity can be gleaned from other parts of this specification aswell as from references cited in this specification, the entire contentsof which are incorporated herein by cross-reference.

It will be appreciated that the following examples have been providedfor the purpose of illustrating preferred embodiments of the presentinvention. Therefore, it would be understood that the present inventionshould not be considered to be limited solely to the features asdescribed in the examples.

EXAMPLES Example 1—Transcriptomic Analysis

Three specimens of the Australian funnel-web spider Hadronyche infensawere milked to exhaust their venom supply and then three days later thespecimens were anesthetised. The paired venom glands were dissected fromeach specimen and immediately placed in TRIzol® reagent (LifeTechnologies). Total RNA was extracted by standard methods, and mRNAenrichment from total RNA was performed using the Oligotex direct mRNAmini kit (Qiagen). RNA quality and concentration was measured using aBioanalyzer 2100 pico chip (Agilent Technologies).

100 ng of mRNA was used to construct a cDNA library using the standardcDNA rapid library preparation and emulsion PCR method (Roche).Sequencing was performed at the Brisbane node of the Australian GenomeResearch Facility using a Roche GS-FLX sequencer. The Raw StandardFlowgram File (.SFF) was processed and low-quality sequences discardedusing a Phred score cut-off of 25 (Pandey et al., 2010). De novoassembly was undertaken using MIRA software (Chevreux et al., 2004)using the following parameters: -GE:not=4 --project=Hinfensa--job=denovo,est,accurate,454 454_SETTINGS -CL:qc=no -AS:mrpc=1-AL:mrs=99,egp=1. The assembled data set was visualised using TABLET(Milne et al., 2010) or Geneious software (Drummond et al., 2011).Consensus sequences were submitted to the Blast2GO software suiteacquire BLAST and functional annotations (Conesa et al., 2005). Signalsequences were determined using the SignalP algorithm (Bendtsen et al.,2004). Putative propeptide cleavage sites were predicted using theresults of a sequence logo analysis of all known spider-toxin precursorsreported on the ArachnoServer database (Herzig et al., 2011). Afterdetermining all processing signals, toxins were classified intosuperfamilies based on their signal sequence and cysteine framework. Onesuperfamily comprised a peptide designated Hi1a and a number oforthologs with a high level of sequence identity with Hi1a. A sequencealignment of the five members of the Hi1a family is shown in FIG. 1.

Example 2—Recombinant Protein Expression

Synthetic genes encoding Hi1a, or derivatives thereof, with codonsoptimised for expression in E. coli, were produced and subcloned into apLiCc vector by GeneArt (as described in Klint et al. 2013). The pLiCcplasmid contains a Lad promoter that is inducible by isopropylβ-D-1-thiogalactopyranoside (IPTG) and a β-lactamase gene for selectionof transformants using ampicillin Hi1a was encoded as aMalE-His₆-MBP-Hi1a fusion protein. MalE is a signal sequence thattargets the expressed fusion protein to the E. coli periplasm where theDsb enzymes that catalyse disulfide-bond formation are located. The His₆tag enables facile purification of the fusion protein by IMACchromatography, while the MBP tag aids protein solubility. A tobaccoetch virus (TEV) protease recognition site was introduced between theMBP and Hi1a-coding regions so that Hi1a could be released from thefusion protein using TEV protease. Cleavage of Hi1a from the fusionprotein results in an additional serine residue at the N-terminus ofHi1a, which is a vestige of the TEV protease cleavage site.

E. coli BL21 (XDE3) cells were transformed with pLiCc vector containinga codon-optimised Hi1a gene. Cultures of this E. coli strain were grownin Luria-Bertani (LB) medium at 30° C., then Hi1a expression was inducedwith 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) atOD₆₀₀=0.8-1.3. Cells were then grown overnight at 16° C., harvested bycentrifugation, and then lysed under constant pressure (27 kpsi) using acell disruptor (TS Series Cell Disrupter, Constant Systems Ltd,Daventry, UK). The lysate was centrifuged at 4° C. using a BeckmanAvanti J-26 centrifuge and then the supernatant was passed over a Ni-NTASuperflow resin (Qiagen) in order to capture the His₆-MBP-Hi1a fusionprotein. The resin was then washed with 10 mM imidazole to removenon-specific binders, then the His₆-MBP-Hi1a protein was eluted with 400mM imidazole. A 30-kDa cut-off centrifugal filter (Millipore) was usedto remove imidazole and concentrate the fusion protein into ˜5 mL of TNbuffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.8). To this solution, 5 mL ofredox buffer (0.6 mM reduced and 0.4 mM oxidised glutathione) was added.Finally, 100 μg of TEV protease was added and the mixture was incubatedat room temperature overnight to release Hi1a from the His₆-MBP fusionprotein. The final yield of recombinant Hi1a is typically about 100-200micrograms per litre of bacterial culture. The recombinant Hi1a containa non-native serine residue at the N-terminus that was added tofacilitate TEV cleavage.

1% trifluoroacetic acid (TFA) was added to solutions containing theproducts of the His₆-MBP-Hi1a cleavage reaction. Hi1a was then isolatedfrom salts, TEV protease, His₆-MBP, and uncleaved His₆-MBP-Hi1a using aShimadzu Prominence high-performance liquid chromatography (HPLC)system. Separation was performed on a semi-preparative C₄ reversed-phase(RP) HPLC column (Phenomenex, C4, 250×10 mm, particle size 10 μm), usinga flow rate of 5 mL/min and an elution gradient of 10-50% solvent B (90%acetonitrile, 10% H20, 0.043% TFA) over 30 min.

The molecular masses of purified peptides were determined using anAPI-2000 ESI-QdQ triple quadrupole mass spectrometer (AppliedBiosystems, Calif., USA).

A RP-HPLC chromatogram of pure recombinant Hi1a is shown in FIG. 2A.Hi1a elutes as three peaks which have the same mass. Re-injection of anyof these single peaks results in the re-appearance of three peaks,indicating that the peptide undergoes conformational isomerism. TheESI-MS trace for pure recombinant Hi1a is shown in FIG. 2B. The averagemass of 8723 Da indicates that the peptide contains six disulfide bonds.

Example 3—Peptide Activity

The ability of the peptides of the invention to modulate ASIC currentswas assessed using two-electrode voltage clamp (TEVC) electrophysiologyexperiments performed on Xenopus laevis oocytes expressing homomericASIC channels. Xenopus oocytes are widely used as an expression systemfor functional characterisation of ion channel modulators. TEVC allowsmeasurement of whole-cell ion channel currents while controlling themembrane potential.

Oocytes were harvested from anaesthetised female X. laevis thendefolliculated by treatment with collagenase (Sigma, type I). Oocyteswere injected with in vitro transcribed ASIC cRNA (capped RNA). Oocyteswere kept at 17-18° C. in ND96 solution containing 96 mM NaCl, 2 mM KCl,1.8 mM CaCl₂, 2 mM MgCl₂, 5 mM HEPES, 5 mM pyruvic acid, 50 μg/mlgentamicin (pH 7.4), and fetal horse serum (2.5%), and experiments wereperformed at room temperature (21-22° C.) 1-3 days after cRNA injection.Oocytes were clamped at −60 mV (Warner OC-725C oocyte clamp; WarnerInstruments, CT, USA) using two standard glass microelectrodes of 0.5-2MΩ resistance when filled with 3 M KCl. Data acquisition and analysiswere performed using pCLAMP software, Version 10 (Axon Instruments, CA,USA). Currents were elicited by a drop in pH from 7.45 to 6.0 using amicroperfusion system to allow rapid solution exchange. All experimentswere performed using ND96 solution spiked with 0.1% BSA in order tominimise adsorption of peptides to plastic tubing.

Concentration effect curves for inhibition of rASIC1a and hASIC1a byHi1a are shown in FIG. 3A (mean±SEM; number of experiments=8; I/I_(max):test current/control current). Fitting of the Hill equation to theconcentration-effect data revealed that Hi1a inhibits rat ASIC1a with ahalf-maximal inhibitory concentration (IC₅₀) of 0.46±0.16 nM and humanASIC1a with an IC₅₀ of 0.59±0.14 nM. The curves also indicate thatinhibition of ASIC1a by Hi1a is incomplete, with ˜30% of rASIC1a and˜20% of hASIC1a currents remaining at saturating concentrations of theHi1a peptide.

The effect of 30 nM Hi1a at pH 7.45 on various ASIC subtypes is shown inFIG. 3B (mean±SEM; number of experiments =5). At this dose, Hi1a had aminimal effect on rASIC1b, rASIC2a and rASIC3, indicating that Hi1a hasat least 50-fold selectivity for ASIC1a over these other subtypes. Theeffect of 1 nM and 1 μM Hi1a at pH 7.45 on various ASIC subtypes isshown in FIG. 3C (mean±SEM; number of experiments =5). At 1 μM, Hi1a hadno effect on homomeric rASIC2a or rASIC3, and it mildly potentiatedrASIC1b, indicating >2000-fold higher potency at ASIC1a over these othersubtypes.

FIG. 3D shows that Hi1a had no activity on an F350A mutant of rASIC1a.The crystal structure of chicken ASIC1 (Jasti et al., 2007) reveals thatresidue F350 is located on an a-helix in an acidic pocket that isimportant for proton gating of the channel Mutation of this Phe residueto Ala abolishes the ability of another spider-venom peptide, PcTx1, toinhibit ASIC1a (Sherwood et al., 2009). Thus, the binding sites forPcTx1 and Hi1a on ASIC1a at least partly overlap.

The inhibition of ASIC1a by PcTx1 is rapidly reversible upon washout ofthe peptide (fully reversible within ˜15 minutes). In contrast, theinhibitory effect of Hi1a at the same concentration (10 nM) is veryslowly reversible with only ˜40% recovery of current amplitude recoveredafter 30 minutes of washout as shown in FIG. 4.

Example 4—Mechanism of Action of Hi1a on ASIC1a

The mechanism of action of Hi1a on ASIC1a was examined using TEVCelectrophysiology. PcTx1 mimics the effect of protonation of ASIC1a,leading to chronic desensitisation at a physiological pH of 7.4(indicated by a parallel shift in the steady-state desensitisation (SSD)curve to more alkaline values with no decrease in the maximum current).The effect of two concentrations of Hi1a on the SSD curves of rat andhuman ASIC1a were assessed and it was determined that Hi1a does notcause a concentration-dependent parallel shift in the SSD curve to morealkaline values. Instead, Hi1a causes a pH-independent decrease in themaximum current (FIGS. 5 and 7), indicating that Hi1a inhibits ASIC1awith a mode of action that differs from that of PcTx1. Unlike PcTx1,Hi1a appears to inhibit activation rather than stabilizing thedesensitised state.

The ability of Hi1a to affect the pH-dependence of ASIC1a activation wasalso assessed (FIGS. 6 and 7). In the presence of Hi1a (applied at pH7.45), the pHs₅₀ of activation is shifted to slightly lower values in aconcentration-dependent manner, for both rASIC1a and hASIC1a. Thissuggests that Hi1a inhibits ASIC1a by increasing the activationthreshold, therefore preventing activation. This effect on activation isthe opposite of that observed with PcTx1. When applied at pH 7.9, PcTx1shifts the activation curve for ASIC1a to more alkaline values, that is,it makes the channel more sensitive to protons (Chen et al., 2005). Thepresent results indicate that Hi1a makes ASIC1a less sensitive toprotons in an insurmountable way (that is, the antagonism is notcompetitive).

Example 5—Structure-Activity Relationships

The structure of Hi1a was determined using heteronuclear NMRspectroscopy. NMR data were acquired at 298 K on a Bruker Avance II+ 900MHz spectrometer equipped with a cryogenically cooled triple-resonanceprobe using a 300 μM sample of ¹³C/¹⁵N-labelled Hi1a dissolved in 20 mM2-(N-morpholino) ethanesulfonic acid (MES) buffer, 0.02% NaN3 and 5%D₂O. Sequence-specific resonance assignments were obtained using acombination of 2D ¹H-¹⁵N-HSQC, 2D ¹H-¹³C-HSQC, 3D HNCACB, 3D CBCA(CO)NH,3D HNCO, and 3D HBHA(CO)NH spectra. Sidechain resonance assignments weremade using a 4D HCC(CO)NH-TOCSY spectrum which has the advantage ofproviding direct sidechain ¹³C-¹H connectivities. All 3D and 4D spectrawere acquired using non-uniform sampling (NUS) and processed usingmaximum entropy reconstruction. Chemical shift assignments for Hi1a havebeen deposited in BioMagResBank (BMRB) under accession code 25848.

Inter-proton distance restraints for structure calculations were derivedfrom¹³C-aliphatic, ¹³C-aromatic and ¹⁵N-edited NOESY-HSQC spectraacquired using uniform sampling with a mixing time of 200 ms. Resonanceassignments, as well as peak picking and integration of NOESY spectra,were achieved manually using SPARKY, then peaklists were assigned and anensemble of structures calculated automatically using the torsion angledynamics package CYANA 3.0. The chemical shift tolerances used forauto-assignment in CYANA were 0.025 ppm and 0.030 ppm for the direct andindirect ¹H dimensions, respectively, and 0.30 ppm for the heteronucleus(¹³C/¹⁵N). Backbone dihedral-angle restraints derived from TALOSchemical shift analysiswere also incorporated into structurecalculations with the restraint range set to twice the estimatedstandard deviation. Disulfide-bond connectivities were determinedunambiguously in the first round of structure calculations andcorresponding disulfide-bond restraints were applied in subsequentcalculations as described previously. CYANA was used to calculate 200structures from random starting conformations, then the 20 conformerswith highest stereochemical quality as judged by MolProbity wereselected to represent the solution structure of Hi1a.

The structure of Hi1a determined using NMR revealed that it is comprisedof two homologous inhibitor cystine knot (ICK) domains connected via ashort and structurally well-defined linker (FIG. 8). Thus, Hi1a is amember of the recently described “double-knot” toxin family Atomiccoordinates for the ensemble of Hi1a structures have been deposited inthe Protein Data Bank (accession number 2N8F).

To determine whether either of the ICK domains of Hi1a are active inisolation, the N-terminal (Hi1a-N) and C-terminal (Hi1a-C) ICK domainswere produced separately by expression in E. coli. Functionalcharacterisation revealed that Hi1a_N can fully inhibit ASIC1a with anIC₅₀ of ˜1 μM (i.e., ˜1000-fold lower potency than either PcTx1 orfull-length Hi1a). Unlike full length Hi1a, this effect was fullyreversible (described below). There was no observed inhibitory effectfor Hi1a_C up to 30 μM concentration (FIG. 9); however, at 1 μM, Hi1a_Cinduced minor potentiation of the current. Three constructs of Hi1a_Nwith varying C-terminal sequences were prepared (SEQ ID NOs:22 to 24).The concentration-effect curves for inhibition of rASIC1a by the threeHi1a_N constructs (FIG. 9) show that modifying the C-terminal sequencedoes not improve the activity of the N-terminal ICK domain of Hi1a.

FIG. 10 shows whole-cell recordings from oocytes expressing rASIC1a whenHi1a-N and Hi1a_C were co-applied or alternatively when Hi1a_N andHi1a_C were applied sequentially, 60 seconds apart. These experimentsrevealed that rASIC1a inhibition by Hi1a_N is fully reversible, whichcan be seen as full recovery of rASIC1a currents 60 seconds afterwashout of the peptide. There was negligible inhibition by Hi1a_C at thechosen concentration (10 μM). Co-application of both peptides, orapplication of one followed 60 seconds later by the other, did not causesignificant inhibition of rASIC1a over that caused by the N-terminaldomain alone. Thus, covalent linkage of the two ICK domains is necessaryfor the unique inhibitory activity of Hi1a.

Example 6—Mutagenesis Studies Hi1a Point Mutants

Mutagenesis studies were undertaken in order to identify key functionalresidues in Hi1a and to determine whether the spatial relationshipbetween the ICK domains (dictated by the linker) is critical for Hi1apotency.

The N-terminal ICK domain of Hi1a is more homologous to PcTx1 than theC-terminal domain, but Hi1a does not contain several key pharmacophoreresidues (K8, W24 and R28) that were identified via mutagenesis of PcTx1and a crystal structure of the PcTx1:chicken ASIC1 complex. To study theeffect that alterations in these key pharmacophore residues has on thepotency of Hi1a, the point mutations L8K, Y24W and H28R were introducedinto Hi1a to make it more closely resemble PcTx1. The activity of thesemutants on rASIC1a is shown in FIG. 11.

Unexpectedly, inserting PcTx1 pharmacophore residues into Hi1a had onlya slight effect on the activity of Hi1a. The mutation H28R led toslightly increased potency (i.e., slightly lower IC₅₀ value) compared towild-type Hi1a while L8K had little effect. Most notably, introductionof a Y24W mutation caused full inhibition of rASIC1a currents whenapplied at saturating concentrations. The effects of these threemutations (L8K, H28R, Y24W) could not have been predicted from previousstudies of PcTx1. All three of the mutant Hi1a peptides appear to bindwith very slow reversibility to rASIC1a, similar to wild-type Hi1a.

Engineered Peptides

Hi1a resembles two concatenated, non-identical copies of an ICK peptidewith homology to PcTx1. In order to understand the unique mechanism ofaction and very slow reversibility of Hi1a, several peptides containingtwo ICK motifs with different linkers were designed and produced in E.coli (SEQ ID NOs:25 to 28). EP1 (SEQ ID NO:25) consists of two copies ofPcTx1 joined by a linker as close to native PcTx1 as possible using thelength of the linker in Hi1a. EP 2 (SEQ ID NO:26) is the same as EP 1but with a Glu to Gly mutation in the linker to remove a negative chargeand increase flexibility (this position is a Gly in the Hi1a linker). EP3 (SEQ ID NO:27) is the same as Hi1a but with a modified linker in whichthe Ile-Pro sequence is replaced by five glycine residues, whilst inPcTx1/Hi1a_C (SEQ ID NO:28) the N-terminal ICK domain of Hi1a isreplaced with PcTx1 and this chimeric peptide has a longer linker.

Functional characterisation of EP 1 and EP 2 revealed that thesepeptides inhibit rASIC1a with IC₅₀ values of 64 nM and 215 nM,respectively (FIG. 12), which corresponds to a ˜200- and 500-foldincrease in IC₅₀ (that is, decrease in potency) when compared towild-type Hi1a (FIG. 2). Both EP 1 and EP 2, like PcTx1, induce fullinhibition of ASIC1a currents. However, EP 3, which consists of the twoICK domains of Hi1a joined by a linker comprised of six glycineresidues, did not inhibit rASIC1a currents by more than ˜20% whenapplied at up to 1 μM. This demonstrates that the linker region iscrucial for the activity of Hi1a. The activity of PcTx1/Hi1a_C issimilar to Hi1a; it induces incomplete inhibition, albeit with 3-folddecrease in potency relative to Hi1a. Interestingly, all threeinhibitory peptides (EP 1, EP 2 and PcTx1/Hi1a_C) appear to bind ASIC1 ain an slowly reversible manner, similar to wild-type Hi1a.

Activity of Hi1d

Hi1d is another member of the Hi1a peptide family found in thevenom-gland transcriptome of Hadronyche infensa. It has the samecysteine scaffold as Hi1a and the sequences of the two peptides are 63%identical. The N-terminal ICK domain of Hi1d is more similar to PcTx1than is the N-terminal ICK domain of Hi1a, and Hi1d has a substantiallydifferent linker between the two ICK domains (8 residues, VPITQKIF, SEQID NO:41) than that of Hi1a (6 residues, VPIPGF, SEQ ID NO:42).Furthermore, there are several substantial differences in the C-terminalICK domain (FIG. 13). To determine the functional significance of thesedifferences, the activity of Hi1d was investigated using TEVC. Hi1dinduced complete, concentration-dependent inhibition of rASIC1 acurrents with an IC₅₀ of 193 nM (FIG. 13). Moreover, the inhibition ofASIC1a by Hi1d was fully reversible within 60 seconds of washout.

A summary of the activity of the peptides tested is provided in Table 1.

TABLE 1 Summary of peptides examined for inhibition of ASIC1a. %Residual Rapidly reversible Peptide IC₅₀ Current inhibition Hi1arASIC1a-460 pM rASIC1a-30% No hASIC1a-590 pM hASIC1a-20% No Hi1a L8K 976pM 30%  No Hi1a Y24W 537 pM 0% No Hi1a H28R 115 pM 20%  No Hi1d 193 nM0% Yes EP 1  64 nM 0% No EP 2 215 nM 0% No EP 3  >4 nM 80%  N/APcTx1/Hi1a_C  1.3 nM ~35%   No

Taken together, these results show that simply linking two identical ICKdomains together is not sufficient to recapitulate the unique inhibitoryactivity of Hi1a at ASIC1a. The results also suggest that either theflexibility or chemical nature of the linker (or both) affects thepeptide's ability to bind to and inhibit ASIC1a channels.

We therefore predict the following general structure for aneuroprotective peptide capable of specifically binding to acid sensingion channel subtype 1a (ASIC1a):

X-L-Y   (I)

wherein X and Y each represent a peptide sequence having an ICK fold andL is a linker.

The linker can be a bond, a non-amino acid-based chemical moiety, asingle amino acid, or a peptide sequence. Where the linker is a singleamino acid, it can be a naturally occurring amino acid or a synthetic,non-naturally occurring amino acid such as a chemical analogue of acorresponding naturally occurring amino acid. Alternatively, the linkercan be a peptide sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 amino acids. The amino acids of the sequence can bethe same natural or non-natural amino acid, or each amino acid of thesequence can be independently selected from a naturally occurring aminoacid or a synthetic, non-naturally occurring amino acid. Preferably, thelinker is a peptide sequence of six naturally occurring amino acids.

X and Y can be different sequences or they can be the same sequence.Preferably, X and Y are different sequences, but both fold to form anICK motif. Each X and Y is therefore preferably a sequence of about 30to about 50 amino acids, even more preferably, about 30, 31, 32, 33, 34,35, 36, 37, 38, 39 or 40 amino acids.

Example 7—Neuroprotection Assays

In vivo—i.c.v. Administration

The neuroprotective efficacy of Hi1a was assessed in a rat model offocal cerebral ischemia. In vivo experiments were conducted using afocal reperfusion model of stroke in conscious spontaneouslyhypertensive rats (SHR) as previously reported (McCarthy et al.,Neuropharmacology 99, 650-657, 2015). Two 23-gauge stainless steel guidecannulae were stereotaxically implanted into anaesthetised animals(ketamine 75 mg/kg; xylazine 10 mg/kg; intraperitoneal) 5 days prior tostroke induction. The first cannula was implanted 3 mm dorsal to theright middle cerebral artery for stroke induction. The second cannulawas implanted into the left lateral ventricle for drug administration.

After 5 days recovery, stroke was induced in conscious rats by insertinga 30-gauge injector protruding 3 mm below the previously implantedcannula. Endothelin-1 (20 pmol/μl) was administered at a rate of 0.2 μlevery 30 s until animals exhibited behaviours previously correlated withstroke severity. Only animals that achieved a level 4 stroke wereincluded in the study. Typical behaviours associated with a level 4stroke include continuous ipsilateral circling, clenching, dragging,failure to extend the left contralateral forelimb, chewing, jaw flexing,and shuffling with forepaws.

Compared to models of middle cerebral artery occlusion (MCAO) achievedby surgical exposure of the artery, this model has the advantage ofminimal surgical intervention in the brain, and the stroke is induced inthe absence of anaesthetics, which can confound the results of drugstudies (Callaway et al., 1999; Sharkey et al., 1993). Stroke-inducedmotor deficit was assessed by counting the number of foot faults madewhile rats traversed a gradually narrowing ledged beam (McCarthy et al.,Stroke 40, 1482-1489, 2009). Animals were trained to traverse the beamon two consecutive days prior to pre-stroke assessment. For theneurological score, postural abnormalities were assessed by elevatingthe rat by the tail above a flat surface and grading the severity ofthorax twisting and the angle of forelimb extension. Both indicators ofneurological health are scored between 0 and 3, with a score of 0corresponding to no twisting of the thorax or complete forelimbextension towards the flat surface. A score of 3 corresponds to severethorax twisting and a failure to extend the forelimb. The score forthorax twisting and forelimb extension are summed to give a totalpossible score of 6, which represents severe neurological deficit. Bothbehavioural tests were performed prior to stroke, and at 24 and 72 hafter stroke.

At 72 h post-stroke, rats were re-anaesthetised (ketamine 75 mg/kg;xylazine 10 mg/kg; intraperitoneal) and transcardially perfused withphysiologically buffered saline. Brains were removed, snap frozen andsectioned (16 μm) at eight pre-determined forebrain levels (−3.20 mm to6.8 mm) relative to bregma. Sections were imaged, and infarct volumeswere measured using the ballistic light method and corrected for edema(McCarthy et al., Stroke 40, 1482-1489, 2009).

In one series of experiments, SHR were treated two hours after strokeinduction with a single intracerebroventricular (i.c.v.) dose of Hi1a (2ng/kg), PcTx1 (1 ng/kg), a double-mutant PcTx1 analogue(R27A/V32A=“PcTx1 mutant iso 2”; McCarthy et al., 2015) that is inactiveagainst ASIC1a (1 ng/kg), or saline (vehicle) using a 30-gauge injectorprotruding 3 mm below the previously implanted guide cannula. The dosesof Hi1a and PcTx1 were chosen to give approximately equivalent molarconcentrations as Hi1a is twice the molecular mass of PcTx1. Drugs weredissolved in saline and infused in a volume of 3 μl over 3 min At 3 daysafter stroke, the brains were perfused, removed, then snap frozen andsectioned to determine the infarct size. The results, with data pointsas mean±standard error of mean (SEM) from experiments with sevenanimals, are shown in FIG. 14. Both PcTx1 and Hi1a caused a significantreduction in infarct volume in both the penumbral (cortical) region andischemic (striatal) core, but Hi1a was more effective than PcTx1. Thedisarmed PcTx1 mutant was completely ineffective at reducing infarctvolume.

In a separate series of experiments, Hi1a (2 ng/kg) was administeredi.c.v. to SHR at 2, 4 or 8 hours after stroke induction.Motor-coordination (ledged beam test) and stroke-related posturalabnormalities (neurological test) were evaluated at days 1 and 3post-stroke. At 3 days after stroke, the brains were perfused, removed,then snap frozen and sectioned to determine the infarct size.

FIG. 15a shows infarct volumes following i.c.v. administration ofvehicle (saline) or Hi1a (2 ng/kg) at 2, 4 or 8 hours post-stroke (ps).A single dose of Hi1a (2 ng/kg) was sufficient to dramatically reduceinfarct size by 63-83%. The coronal sections in FIG. 15b show typicalinfarcted and non-infarcted regions from SHR treated i.c.v. with eithervehicle or Hi1a (2 ng/kg) 8 hours after stroke. Notably, Hi1a caused areduction in infarct volume in the ischemic (striatal) core as well asthe penumbral region (cortical or peri-infarct zone) at all time points.

The reduction in infarct size caused by Hi1a administration wasreflected symptomatically, with Hi1a-treated animals exhibiting markedlyreduced neurological deficit (FIG. 16a ) and motor impairment (FIGS. 16b), and it is also correlated with preservation of neuronal architecturein both the penumbral and core regions of damage, as evidenced by intactneuronal staining as shown in FIGS. 17a and 17 b.

In Vivo—Intranasal Administration

Four hours after stroke induction, SHR were treated with a singleintranasal (i.n.) dose of Hi1a (2 μg/kg) or vehicle (saline). The Hi1aor vehicle was delivered i.n. in 3 μL delivered to the left nostril viaa soft catheter inserted 6 mm into the nostril. Motor-coordination(ledged beam test) and stroke-related postural abnormalities(neurological test) were evaluated at 24 and 72 hours post-stroke. At 72hours after stroke, the brains were perfused, removed, then snap frozenand sectioned to determine infarct size. The results are shown in FIG.18.

Intranasal administration of Hi1a four hours after stroke inductiondramatically reduced infarct size. Hi1a afforded protection not only inthe cortical zone (FIG. 18A), but also in the striatal core (FIG. 18B).The preservation of brain tissue was also reflected symptomatically,with Hi1a-treated animals experiencing less motor impairment as judgedby a ledged beam assay (FIG. 18C), reduced neurological deficit (FIG.18D), and suffering less weight loss after stroke than untreated rats(FIG. 18E).

Therapeutic Uses

Acidosis occurs when the extracellular pH falls below 7.35. This is acommon event in many pathophysiological conditions that affect thecentral and/or peripheral nervous system. Tissue injury (e.g., incisionsor trauma), inflammation, arthritis, infection, ischemia, exercise andcancer all cause the local pH to decrease sufficiently (<7) to activatehomomeric ASIC1a (Dube et al., 2009). In both rodents and humans, ASIC1ais highly expressed in sensory and central neurons, which makes themideally placed to sense the acidosis associated with these conditions.Furthermore, the function of ASIC1a is substantially enhanced byextracellular mediators that are released during periods of ischaemiaand inflammation (Deval and Lingueglia, 2015; Huang et al., 2015). Theseproperties of ASIC1a makes this channel a potential drug target forseveral neurodegenerative and neuroinflammatory diseases.

Following an experimentally-induced traumatic injury in mice, the braincortex becomes acidic, as observed in humans following brain trauma. Theresulting acidosis activates ASIC1a, which contributes to secondary celldamage and neurological deficits. Genetic knockout of ASIC1a leads toreduced neurodegeneration after traumatic brain injury (Yin et al.,2013). Inhibition of ASIC1a using Hi1a may therefore provide a noveltherapeutic approach to reduce neurological deficits following traumaticbrain injury.

Epilepsy is a neurological disorder caused by hyperactivity of brainneurons. Even though the onset of seizures (and the ion channelsinvolved) has been extensively studied, the molecular mechanism by whichseizures are terminated remains unclear. It has been shown thatexcessive firing of action potentials reduces brain pH, which in turnhalts the seizure (Somjen, 1984). Disrupting ASIC1a in mice causes moresevere seizures, while overexpressing ASIC1a does not affect seizureonset, but shortens their duration (Ziemann et al., 2008). These datasuggest that acidosis, and ASIC1a activation, limits seizure-likeactivity in the brain, and therefore identifies ASIC1a as a potentialmarker of seizure termination, opening new perspectives in the design ofanti-epileptic drugs.

Huntington's disease (HD) is an autosomal dominant neurological disorderrelated to the expansion of a CAG repeat in the HD gene. This expansioncauses misfolding of its gene product, huntingtin protein, resulting inmuscle spasms and dementia. Aggregation of misfolded huntingtin in braincells causes the formation of inclusion bodies, which disrupts normalneurotransmitters and eventually overall neuronal trafficking(Rubinsztein and Carmichael, 2003). HD is associated with acidificationsufficient to activate ASIC1a in the CNS, which might contribute to thepathogenesis and formation of aggregates. Suppressing the expression ofASIC1a or its pharmacological inhibition in vivo enhances the activityof a specific proteasome, which reduces aggregation of huntingtin (Wonget al., 2008). Thus, ASIC1a is a potential new drug target for thisdebilitating disease.

Multiple sclerosis (MS) is an autoimmune disease in which the myelinsheath surrounding axons of the brain and spinal cord is damaged. Thisneuroinflammatory insult leads to axonal degeneration which in turnleads to progressive motor, sensory, and cognitive disabilities(Filippi, 2011). A recent study on both animal models and human tissueidentified an increased level of ASIC1a expression in glial cells andaxons from MS lesions (Vergo et al., 2011). Furthermore, ASIC1^(−/−)mice were found to present symptoms of much lower severity in models ofMS. These findings suggest that activation of ASIC1a can underlie theneurodegeneration seen in MS and that ASIC1a inhibitors have thepotential to provide both neuro- and myelo-protective benefits in MS.

Spinal cord injury leads to local acidosis from tissue trauma andischaemia, which results in secondary cell death. ASIC1a isover-expressed in injured spinal cord nerves and support cells, and ithas been shown to play a substantial role in the secondary damage andresulting loss of function (Hu et al., 2011). Inhibition of ASIC1a isneuroprotective in spinal cord injury (Hu et al., 2011; Koehn et al.,F1000Research, 2016), and therefore novel inhibitors of ASIC1a such asHi1a have potential for treatment of spinal cord injury.

It is to be appreciated that conditions other than those described abovemay be caused by ASIC1a activity or contributed to by ASIC1a activity,and so the peptide described by formula (I) may be used in theprophylaxis or treatment of those other conditions. That is, the peptidemay function therapeutically other than as a neuroprotective agent.

Routes of Administration and Doses

ASIC1a is a therapeutic target with a temporal window that is in keepingwith the clinical presentation after an ischemic event. Pure Hi1apeptide yielded excellent neuroprotection of both the striatal (core)and cortical (peri-infarct) region in rats when administered via eitheri.c.v. injection or intranasal administration up to 4 hours afterinduction of stroke. Therefore i.c.v. or intranasal administration maybe used as route of administration.

Under normal circumstances the entry of ions, small molecules andproteins to the brain is tightly regulated by the blood brain barrier(BBB). However, pathological insults to the brain, such as stroke ortraumatic brain injury, result in a rapid increase in the permeabilityof the BBB. Following traumatic brain injury molecules from 286 to10,000 Da (Hi1a is ˜8700 Da) can enter the brain freely for up to fourdays (Habgood et al., 2007). Similarly, following ischemia there is abiphasic opening of the BBB (mediated by the activation of differentgroups of metalloproteinases) commencing within hours of insult andlasting up to 3 days (Yang and Rosenberg, 2011). Thus, in a clinicalpost-stroke setting, in the presence of a compromised BBB, intravenous(i.v.) administration of Hi1a may result in neuroprotection.

In the preferred case, a method of peptide drug delivery that bypasseshepatic metabolism and the BBB entirely could be used. Intranasal (i.n.)administration is non-invasive, leads to uptake directly to the brain,and it avoids both hepatic metabolism and the BBB. There are severalintranasally administered peptide drugs already on the market (Casettariand Ilium, 2014). Intranasal delivery of PcTx1 crude venom waspreviously shown to provide neuroprotection up to 4 hours post-stroke(Pignataro et al., 2007), and we have demonstrated that intranasaldelivery of Hi1a alone up to at least four hours post-stroke provideshigh levels of neuroprotection (FIG. 18). Thus, in a clinicalpost-stroke setting, therapeutic application of Hi1a and modificationsthereof could be achieved via i.c.v., i.n., or i.v. administration.

i.c.v. administration of a functionally-relevant dose of pure Hi1a inconscious SHR rats is highly neuroprotective when given post-stroke. Thedose of Hi1a used in the example (2 ng/kg i.c.v.) equates to a brainconcentration of ˜1.2 nM, which should inhibit brain ASIC1a activity by˜80% based on in vitro data. Thus i.c.v. dosing would preferably be inthe range of 1-10 ng/kg.

Reference throughout this specification to ‘one embodiment’ or ‘anembodiment’ means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearance of the phrases ‘in one embodiment’ or ‘in an embodiment’ invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more combinations.

In compliance with the statute, the invention has been described inlanguage more or less specific to structural or methodical features. Itis to be understood that the invention is not limited to specificfeatures shown or described since the means herein described comprisespreferred forms of putting the invention into effect. The invention is,therefore, claimed in any of its forms or modifications within theproper scope of the appended claims (if any) appropriately interpretedby those skilled in the art.

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1-98. (canceled)
 99. An isolated, synthetic, or recombinant peptide,wherein the peptide comprises an amino acid sequence consisting of an Nterminal serine residue and any one of SEQ ID NOs: 18, 19, and
 20. 100.The isolated, synthetic, or recombinant peptide of claim 99, wherein thepeptide consists of a) an amino acid sequence consisting of an Nterminal serine residue and any one of SEQ ID NOs: 18, 19, and 20, or b)SEQ ID NO:
 17. 101. A pharmaceutical composition comprising atherapeutically effective amount of the isolated, synthetic, orrecombinant peptide of claim
 99. 102. The pharmaceutical composition ofclaim 101, further comprising at least one pharmaceutically acceptablediluent, adjuvant, excipient, buffer, stabiliser, isotonicising agent,preservative, or anti-oxidant.
 103. The pharmaceutical composition ofclaim 102, further comprising at least one delivery enhancer to assistwith transport of the peptide across the blood brain barrier (BBB),wherein the at least one delivery enhancer is selected from the groupconsisting of lipids, polymers, liposomes, emulsions, and nanoparticles.104. A method for treatment of a disease or condition in a subject,wherein the disease or condition is caused by ASIC1a activity orcontributed by ASIC1a activity, the method comprising: administering atherapeutically effective amount of at least one isolated, synthetic, orrecombinant peptide to the subject, wherein the peptide comprises anamino acid sequence selected from the group consisting of: (a) an aminoacid sequence consisting of an N terminal serine residue and any one ofSEQ ID NOs: 18, 19, and 20; (b) the amino acid sequence of SEQ ID NO:17; (c) an amino acid sequence consisting of an N terminal serineresidue and an amino acid sequence that shares at least 90% sequenceidentity with any one of SEQ ID NOs: 18, 19, and 20; and (d) an aminoacid sequence that shares at least 90% sequence identity with SEQ ID NO:17, wherein the amino acid sequence comprises six cysteine residuescovalently joined in pairs to form three disulfide bonds, such that theamino acid sequence comprises one ICK motif, and wherein said peptide iscapable of specifically binding to acid sensing ion channel subtype 1a(ASIC1a).
 105. The method of claim 104, wherein the isolated, syntheticor recombinant peptide comprises (a) an amino acid sequence consistingof an N terminal serine residue and any one of SEQ ID NOs: 18, 19, and20; or (b) SEQ ID NO:
 17. 106. The method of claim 104, wherein thepeptide comprises (a) an amino acid sequence consisting of an N terminalserine residue and the amino acid sequence of SEQ ID NO: 19; or (b) SEQID NO:
 17. 107. The method of claim 104, wherein the peptide comprisesan amino acid sequence consisting of an N terminal serine residue andthe amino acid sequence of SEQ ID NO: 18 or SEQ ID NO:
 20. 108. Themethod of claim 104, wherein the disease or condition caused by ASIC1aactivity or contributed to by ASIC1a activity is an ischemic disease.109. The method of claim 104, wherein the disease or condition isstroke.
 110. The method of claim 104, wherein the disease or conditionis neuronal damage following stroke.
 111. The method of claim 104,wherein the isolated, synthetic or recombinant peptide is administeredvia intracerebroventricular (i.c.v.) administration, intravenous (i.v.)administration, intraarterial (i.a.), or intranasal (i.n.)administration.
 112. The method of claim 108, wherein the ischemicdisease is cardiac ischemia.
 113. The method of claim 104, wherein thedisease or condition is angina associated with cardiac ischemia. 114.The method of claim 104, wherein the disease or condition is spinal cordinjury.
 115. The method of claim 114, wherein the spinal cord injury isassociated with multiple sclerosis (MS).
 116. The method of claim 104,wherein administering the peptide comprises administering apharmaceutical composition comprising the peptide.
 117. The method ofclaim 116, wherein the pharmaceutical composition comprises at least onepharmaceutically acceptable diluent, adjuvant, excipient, buffer,stabiliser, isotonicising agent, preservative, or anti-oxidant.
 118. Themethod of claim 117, wherein the pharmaceutical composition furthercomprises at least one delivery enhancer to assist with transport of thepeptide across the blood brain barrier (BBB), wherein the at least onedelivery enhancer is selected from the group consisting of lipids,polymers, liposomes, emulsions, and nanoparticles.